Modified enzymes having polymer conjugates

[0002] This application is a divisional of application Ser. No. 09/024,532 filed on Feb. 17, 1998, now U.S. Pat. No. 6,245,901, which is a continuation of PCT/DK98/00046 filed on Feb. 6, 1998, and claims priority under 35 U.S.C. 119 of Danish application no. 0135/97 filed Feb. 6, 1997, the contents of which are fully incorporated herein by reference.

[0003] The present invention relates to polypeptide-polymer conjugates having added and/or removed one or more attachment groups for coupling polymeric molecules on the surface of the 3D structure of the polypeptide, a method for preparing polypeptide-polymer conjugates of the invention, the use of said conjugated for reducing the immunogenicity and allergenicity, and compositions comprising said conjugate.

[0004] The use of polypeptides, including enzymes, in the circulatory system to obtain a particular physiological effect is well-known in the medical arts. Further, within the arts of industrial applications, such as laundry washing, textile bleaching, person care, contact lens cleaning, food and feed preparation enzymes are used as a functional ingredient. One of the important differences between pharmaceutical and industrial application is that for the latter type of applications (i.e. industrial applications) the polypeptides (often enzymes) are not intended to enter into the circulatory system of the body.

[0005] Certain polypeptides and enzymes have an unsatisfactory stability and may under certain circumstances—dependent on the way of challenge—cause an immune response, typically an IgG and/or IgE response.

[0006] It is today generally recognized that the stability of polypeptides is improved and the immune response is reduced when polypeptides, such as enzymes, are coupled to polymeric molecules. It is believed that the reduced immune response is a result of the shielding of (the) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation by the coupled polymeric molecules.

[0007] Techniques for conjugating polymeric molecules to polypeptides are well-known in the art.

[0008] One of the first commercially suitable techniques was described back in the early 1970's and disclosed in e.g. U.S. Pat. No. 4,179,337. Said patent concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG). At least 15% of polypeptides' physiological activity is maintained.

[0009] GB patent no. 1,183,257 (Crook et al.) describes chemistry for conjugation of enzymes to polysaccharides via a triazine ring.

[0010] Further, techniques for maintaining of the enzymatic activity of enzyme-polymer conjugates are also known in the art.

[0011] WO 93/15189 (Veronese et al.) concerns a method for maintaining the activity in polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a macromolecularized inhibitor. The conjugates are intended for medical applications.

[0012] It has been found that the attachment of polymeric molecules to a polypeptide often has the effect of reducing the activity of the polypeptide by interfering with the interaction between the polypeptide and its substrate. EP 183 503 (Beecham Group PLC) discloses a development of the above concept by providing conjugates comprising pharmaceutically useful proteins linked to at least one water-soluble polymer by means of a reversible linking group.

[0013] EP 471,125 (Kanebo) discloses skin care products comprising a parent protease (Bacillus protease with the trade name Esperase®) coupled to polysaccharides through a triazine ring to improve the thermal and preservation stability. The coupling technique used is also described in the above mentioned GB patent no. 1,183,257 (Crook et al.).

[0014] JP 3083908 describes a skin cosmetic material which contains a transglutaminase from guinea pig liver modified with one or more water-soluble, substances such as PEG, starch, cellulose etc. The modification is performed by activating the polymeric molecules and coupling them to the enzyme. The composition is stated to be mild to the skin.

[0015] However, it is not always possible to readily couple polymeric molecules to polypeptides and enzymes. Further, there is still a need for polypeptide-polymer conjugates with an even more reduced immunogenicity and/or allergenicity.

[0016] It is the object of the present invention to provide improved polypeptide-polymer conjugates suitable for industrial and pharmaceutical applications.

[0017] The term “improved polypeptide-polymer conjugates” means in the context of the present invention conjugates having a reduced immune response in humans and animals and/or a improved stability. As will be described further below the immune response is dependent on the way of challenge.

[0018] The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or allergenic by adding and/or removing one or more attachment groups on the surface of the parent polypeptide to be coupled to polymeric molecules.

[0019] When introducing pharmaceutical polypeptide directly into the circulatory system (i.e. bloodstream) the potential risk is an immunogenic response in the form of mainly IgG, IgA and/or IgM antibodies. In contrast hereto, industrial polypeptides, such as enzymes used as a functional ingredient in e.g. detergents, are not intended to enter the circulatory system. The potential risk in connection with industrial polypeptides is inhalation causing an allergenic response in the form of mainly IgE antibody formation.

[0020] Therefore, in connection with industrial polypeptides the potential risk is respiratory allergenicity caused by inhalation, intratracheal and intranasal presentation of polypeptides.

[0021] The main potential risk of pharmaceutical polypeptides is immunogenicity caused by, intradermal, intravenous, or subcutaneous presentation of the polypeptide.

[0022] It is to be understood that reducing the “immunogenicity” and reducing the “respiratory allergenicity” are two very different problems based on different routes of exposure and on two very different immunological mechanisms:

[0023] The term “immunogenicity” used in connection with the present invention may be referred to as allergic contact dermatitis in a clinical setting and is a cell mediated delayed immune response to chemicals that contact and penetrate the skin. This cell mediated reaction is also termed delayed contact hypersensitivity (type IV reaction according to Gell and Combs classification of immune mechanisms in tissue damage).

[0024] The term “allergenicity” or “respiratory allergenicity” is an immediate anaphylactic reaction (type I antibody-mediated reaction according to Gell and Combs) following inhalation of e.g. polypeptides.

[0025] According to the present invention it is possible to provide polypeptides with a reduced immune response and/or improved stability, which has a substantially retained residual activity.

[0026] The allergic and the immunogenic response are in one term, at least in the context of the present invention called the “immune response”.

[0027] In the first aspect the invention relates to a polypeptide-polymer conjugate having

[0028] a) one or more additional polymeric molecules coupled to the polypeptide having been modified in a manner to increase the number of attachment groups on the surface of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide, and/or

[0029] b) one or more fewer polymeric molecules coupled to the polypeptide having been modified in a manner to decrease the number of attachment groups at or close to the functional site(s) of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide.

[0030] The term “parent polypeptide” refers to the polypeptide to be modified by coupling to polymeric molecules. The parent polypeptide may be a naturally-occurring (or wild-type) polypeptide or may be a variant thereof prepared by any suitable means. For instance, the parent polypeptide may be a variant of a naturally-occurring polypeptide which has been modified by substitution, deletion or truncation of one or more amino acid residues or by addition or insertion of one or more amino acid residues to the amino acid sequence of a naturally-occurring polypeptide.

[0031] A “suitable attachment group” means in the context of the present invention any amino acid residue group on the surface of the polypeptide capable of coupling to the polymeric molecule in question.

[0032] Preferred attachment groups are amino groups of Lysine residues and the N-terminal amino group. Polymeric molecules may also be coupled to the carboxylic acid groups (—COOH) of amino acid residues in the polypeptide chain located on the surface. Carboxylic acid attachment groups may be the carboxylic acid group of Aspartate or Glutamate and the C-terminal COOH-group.

[0033] A “functional site” means any amino acid residues and/or cofactors which are known to be essential for the performance of the polypeptide, such as catalytic activity, e.g. the catalytic triad residues, Histidine, Aspartate and Serine in Serine proteases, or e.g. the heme group and the distal and proximal Histidines in a peroxidase such as the Arthromyces ramosusperoxidase.

[0034] In the second aspect the invention relates to a method for preparing improved polypeptide-polymer conjugates comprising the steps of:

[0035] a) identifying amino acid residues located on the surface of the 3D structure of the parent polypeptide in question,

[0036] b) selecting target amino acid residues on the surface of said 3D structure of said parent polypeptide to be mutated,

[0037] c) i) substituting or inserting one or more amino acid residues selected in step b) with an amino acid residue having a suitable attachment group, and/or ii) substituting or deleting one or more amino acid residues selected in step b) at or close to the functional site(s),

[0038] d) coupling polymeric molecules to the mutated polypeptide.

[0039] The invention also relates to the use of a conjugate of the invention and the method of the invention for reducing the immunogenicity of pharmaceuticals and reducing the allergenicity of industrial products.

[0040] Finally the invention relates to compositions comprising a conjugate of the invention and further ingredients used in industrial products or pharmaceuticals.

[0041] FIG. 1 shows the anti-lipase serum antibody levels after 5 weekly immunizations with i) control ii) unmodified lipase variant, iii) lipase variant-SPEG. (X: log(serum dilution); Y optical Density (490/620)).

[0042] It is the object of the present invention to provide improved polypeptide-polymer conjugates suitable for industrial and pharmaceutical applications.

[0043] Even though polypeptides used for pharmaceutical applications and industrial application can be quite different the principle of the present invention may be tailored to the specific type of parent polypeptide (i.e. enzyme, hormone peptides etc.).

[0044] The inventors of the present invention have provided improved polypeptide-polymer conjugates with a reduced immune response in comparison to conjugates prepared from the corresponding parent polypeptides.

[0045] The present inventors have found that polypeptides, such as enzymes, may be made less immunogenic and/or less allergenic by adding one or more attachment groups on the surface of the parent polypeptide. In addition thereto the inventors have found that a higher percentage of maintained residual functional activity may be obtained by removing attachment groups at or close to the functional site(s).

[0046] In the first aspect the invention relates to an improved polypeptide-polymer conjugate having

[0047] a) one or more additional polymeric molecules coupled to the polypeptide having been modified in a manner to increase the number of attachment groups on the surface of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide, and/or

[0048] b) one or more fewer polymeric molecules coupled to the polypeptide having been modified in a manner to decrease the number of attachment groups at or close to the functional site(s) of the polypeptide in comparison to the number of attachment groups available on the corresponding parent polypeptide.

[0049] Whether the attachment groups should be added and/or removed depends on the specific parent polypeptide.

[0050] There may be a need for further attachment groups on the polypeptide if only few attachment groups are available on the surface of the parent polypeptide. The addition of one or more attachment groups by substituting or inserting one or more amino acid residues on the surface of the parent polypeptide increases the number of polymeric molecules which may be attached in comparison to the corresponding parent polypeptide. Conjugates with an increased number of polymeric molecules attached thereto are generally seen to have a reduced immune response in comparison to the corresponding conjugates having fewer polymeric molecules coupled thereto.

[0051] Any available amino acid residues on the surface of the polypeptide, preferentially not being at or close to the functional site(s), such as the active site(s) of enzymes, may in principle be subject to substitution and/or insertion to provide additional attachment groups.

[0052] As will be described further below the location of the additional coupled polymeric molecules may be of importance for the reduction of the immune response and the percentage of maintained residual functional activity of the polypeptide itself.

[0053] A conjugate of the invention may typically have from 1 to 25, preferentially 1 to 10 or more additional polymeric molecules coupled to the surface of the polypeptide in comparison to the number of polymeric molecules of a conjugate prepared on the basis of the corresponding parent polypeptide.

[0054] However, the optimal number of attachment groups to be added depends (at least partly) on the surface area (i.e. molecular weight) of the parent polypeptide to be shielded by the coupled polymeric molecules, and also on the number of already available attachment groups on the parent polypeptide.

[0055] In the case of enzymes or other polypeptides performing their function by interaction with a substrate or the like, polymeric molecules coupled to the polypeptide might be impeded by the interaction between the polypeptide and its substrate or the like, if they are coupled at or close to the functional site(s) (i.e. active site of enzymes). This will most probably cause reduced activity.

[0056] In the case of enzymes having one or more polymeric molecules coupled at or close to the active site a substantial loss of residual enzymatic activity can be expected. Therefore, according to the invention conjugates may be constructed to maintain a higher percentage of residual enzymatic activity in comparison to a corresponding conjugates prepared on the basis of the parent enzyme in question. This may be done by substituting and/or deleting attachment groups at or close to the active site, hereby increasing the substrate affinity by improving the accessibility of the substrate in the catalytic cleft.

[0057] An enzyme-polymer conjugate of the invention may typically have from 1 to 25, preferably 1 to 10 fewer polymeric molecules coupled at or close to the active site in comparison to the number of polymeric molecules of a conjugate prepared on the basis of the corresponding parent polypeptide.

[0058] As will be explained below “at or close to” the functional site(s) means that no polymeric molecule(s) should be coupled within 5 Å, preferably 8 Å, especially 10 Å of the functional site(s).

[0059] Removal of attachment groups at or close to the functional site(s) of the polypeptide may advantageously be combined with addition of attachment groups in other parts of the surface of the polypeptide.

[0060] The total number of attachment groups may this way be unchanged, increased or decreased. However the location(s) of the total number of attachment group(s) is (are) improved assessed by the reduction of the immune response and/or percentage of maintained residual activity. Improved stability may also be obtained this way.

[0061] The Number of Attachment Groups

[0062] Generally seen the number of attachment groups should be balanced to the molecular weight and/or surface area of the polypeptide. The more heavy the polypeptide is the more polymeric molecules should be coupled to the polypeptide to obtain sufficient shielding of the epitope(s) responsible for antibody formation.

[0063] Therefore, if the parent polypeptide molecule is relatively light (e.g. 1 to 35 kDa) it may be advantageous to increase the total number of coupled polymeric molecules (outside the functional site(s)) to a total between 4 and 20.

[0064] If the parent polypeptide molecules is heavier, for instance 35 to 60 kDa, the number of coupled polymeric molecules (outside the functional site(s)) may advantageously be increased to 7 to 40, and so on.

[0065] The ratio between the molecular weight (Mw) of the polypeptide in question and the number of coupled polymeric molecules considered to be suitable by the inventors is listed below in Table 1.

[0066] Reduced Immune Response vs. Maintained Residual Enzymatic Activity

[0067] Especially for enzymes, in comparison to many other types of polypeptides, there is a conflict between reducing the immune response and maintaining a substantial residual enzymatic activity as the activity of enzymes are connected with interaction between a substrate and the active site often present as a cleft in the enzyme structure.

[0068] Without being limited to any theory it is believed that the loss of enzymatic activity of enzyme-polymer conjugates might be a consequence of impeded access of the substrate to the active site in the form of spatial hindrance of the substrate by especially bulky and/or heavy polymeric molecules to the catalytic cleft. It might also, at least partly, be caused by disadvantageous minor structural changes of the 3D structure of the enzyme due to the stress made by the coupling of the polymeric molecules.

[0069] Maintained Residual Activity

[0070] A polypeptide-polymer conjugates of the invention has a substantially maintained functional activity.

[0071] A “substantially” maintained functional activity is in the context of the present invention defined as an activity which is at least between 20% and 30%, preferably between 30% and 40%, more preferably between 40% and 60%, better from 60% up to 80%, even better from 80% up to about 100%, in comparison to the activity of the conjugates prepared on the basis of corresponding parent polypeptides.

[0072] In the case of polypeptide-polymer conjugates of the invention where no polymeric molecules are coupled at or close to the functional site(s) the residual activity may even be up to 100% or very close thereto. If attachment group(s) of the parent polypeptide is(are) removed from the functional site the activity might even be more than 100% in comparison to modified (i.e. polymer coupled) parent polypeptide conjugate.

[0073] Position of Coupled Polymeric Molecules

[0074] To obtain an optimally reduced immune response (i.e. immunogenic and allergenic response) the polymeric molecules coupled to the surface of the polypeptide in question should be located in a suitable distance from each other.

[0075] In a preferred embodiment of the invention the parent polypeptide is modified in a manner whereby the polymeric molecules are spread broadly over the surface of the polypeptide. In the case of the polypeptide in question has enzymatic activity it is preferred to have as few as possible, especially none, polymeric molecules coupled at or close to the area of the active site.

[0076] In the present context “spread broadly over the surface of the polypeptide” means that the available attachment groups are located so that the polymeric molecules shield different parts of the surface, preferably the whole or close to the whole surface area away from the functional site(s), to make sure that epitope(s) are shielded and hereby not recognized by the immune system or its antibodies.

[0077] The area of antibody-polypeptide interaction typically covers an area of 500 Ų, as described by Sheriff et al. (1987), Proc. Natl. Acad. Sci. USA 84, p. 8075-8079. 500 Ųcorresponds to a rectangular box of 25 Å×20 Å or a circular region of radius 12.6 Å. Therefore, to prevent binding of antibodies to the epitope(s) to the polypeptide in question it is preferred to have a maximum distance between two attachment groups around 10 Å.

[0078] Consequently, amino acid residues which are located in excess of 10 Å away from already available attachment groups are suitable target residues. If two or more attachment groups on the polypeptide are located very close to each other it will in most cases result in that only one polymeric molecule will be coupled.

[0079] To ensure a minimal loss of functional activity it is preferred not to couple polymeric molecules at or close to the functional site(s). Said distance depends at least partly on the bulkiness of the polymeric molecules to be coupled, as impeded access by the bulky polymeric molecules to the functional site is undesired. Therefore, the more bulky the polymeric molecules are the longer should the distance from the functional site to the coupled polymeric molecules be.

[0080] To maintain a substantial functional activity of the polypeptide in question attachment groups located within 5 Å, preferred 8 Å, especially 10 Å from such functional site(s) should be left uncoupled and may therefore advantageously be removed or changed by mutation. Functional residues should normally not be mutated/removed, even though they potentially can be the target for coupling polymeric molecules. In said case it may thus be advantageous to choose a coupling chemistry involving different attachment groups.

[0081] Further, to provide a polypeptide having coupled polymeric molecules at (a) known epitope(s) recognizable by the immune system or close to said epitope(s) specific mutations at such sites are also considered advantageous according to the invention. If the position of the epitope(s) is(are) unknown it is advantageous to couple several or many polymeric molecules to the polypeptide.

[0082] As also mentioned above it is preferred that said attachment groups are spread broadly over the surface.

[0083] The Attachment Group

[0084] Virtually all ionized groups, such as the amino groups of Lysine residues, are located on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, (1993), “Proteins”, W. H. Freeman and Company, New York).

[0085] Therefore, the number of readily accessible attachment groups (e.g. amino groups) on a modified or parent polypeptide equals generally seen the number of Lysine residues in the primary structure of the polypeptide plus the N-terminus amino group.

[0086] The chemistry of coupling polymeric molecules to amino groups are quite simple and well established in the art. Therefore, it is preferred to add and/or remove Lysine residues (i.e. attachment groups) to/from the parent polypeptide in question to obtain improved conjugates with reduced immunogenicity and/or allergenicity and/or improved stability and/or high percentage maintained functional activity.

[0087] Polymeric molecules may also be coupled to the carboxylic groups (—COOH) of amino acid residues on the surface of the polypeptide. Therefore, if using carboxylic groups (including the C-terminal group) as attachment groups addition and/or removal of Aspartate and Glutamate residues may also be suitable according to the invention.

[0088] If using other attachment groups, such as —SH groups, they may be added and/or removed analogously.

[0089] Substitution of the amino acid residues is preferred over insertion, as the impact on the 3D structure of the polypeptide normally will be less pronounced.

[0090] Preferred substitutions are conservative substitutions. In the case of increasing the number of attachment groups the substitution may advantageously be performed at a location having a distance of 5 Å, preferred 8 Å, especially 10 Å from the functional site(s) (active site for enzymes).

[0091] An example of a suitable conservative substitution to obtain an additional amino attachment group is an Arginine to Lysine substitution. Examples of conservative substitutions to obtain additional carboxylic attachment groups are Aspargine to Aspartate/Glutamate or Glutamine to Aspartate/Glutamate substitutions. To remove attachment groups a Lysine residue may be substituted with an Arginine and so on.

[0092] The Parent Polypeptide

[0093] In the context of the present invention the term “polypeptides” includes proteins, peptides and/or enzymes for pharmaceutical or industrial applications. Typically the polypeptides in question have a molecular weight in the range between about 1 to 100 kDa, often 15 kDa and 100 kDa.

[0094] Pharmaceutical Polypeptides

[0095] The term “pharmaceutical polypeptides” is defined as polypeptides, including peptides, such as peptide hormones, proteins and/or enzymes, being physiologically active when introduced into the circulatory system of the body of humans and/or animals.

[0096] Pharmaceutical polypeptides are potentially immunogenic as they are introduced into the circulatory system.

[0097] Examples of “pharmaceutical polypeptides” contemplated according to the invention include insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO) and prolactin.

[0098] Industrial Polypeptides

[0099] Polypeptides used for industrial applications often have an enzymatic activity. Industrial polypeptides (e.g. enzymes) are (in contrast to pharmaceutical polypeptides) not intended to be introduced into the circulatory system of the body.

[0100] It is not very like that industrial polypeptides, such as enzymes used as ingredients in industrial compositions and/or products, such as detergents and personal care products, including cosmetics, come into direct contact with the circulatory system of the body of humans or animals, as such enzymes (or products comprising such enzymes) are not injected (or the like) into the bloodstream.

[0101] Therefore, in the case of the industrial polypeptide the potential risk is respiratory allergy (i.e. IgE response) as a consequence of inhalation to polypeptides through the respiratory passage.

[0102] In the context of the present invention “industrial polypeptides” are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be introduced into the circulatory system of the body of humans and/or animals.

[0103] Examples of such polypeptides are polypeptides, especially enzymes, used in products such as detergents, household article products, agrochemicals, personal care products, such as skin care products, including cosmetics and toiletries, oral and dermal pharmaceuticals, composition use for processing textiles, compositions for hard surface cleaning, and compositions used for manufacturing food and feed etc.

[0104] Enzymatic Activity

[0105] Pharmaceutical or industrial polypeptides exhibiting enzymatic activity will often belong to one of the following groups of enzymes including Oxidoreductases (E.C. 1, “Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD); Transferases, (E.C. 2), such as transglutaminases (TGases); Hydrolases (E.C. 3), including proteases, especially subtilisins, and lipolytic enzymes; Isomerases (E.C. 5), such as Protein disulfide Isomerases (PDI).

[0106] Proteolytic Enzymes

[0107] Contemplated proteolytic enzymes include proteases selected from the group of Aspartic proteases, such pepsins, Cysteine proteases, such as Papain, Serine proteases, such as subtilisins, or metallo proteases, such as NEUTRASE®.

[0108] Specific examples of parent proteases include PD498 (WO 93/24623 and SEQ ID NO. 2), SAVINASE® (von der Osten et al., (1993), Journal of Biotechnology, 28, p. 55+, SEQ ID NO 3), Proteinase K (Gunkel et al., (1989), Eur. J. Biochem, 179, p. 185-194), Proteinase R (Samal et al, (1990), Mol. Microbiol, 4, p. 1789-1792), Proteinase T (Samal et al., (1989), Gene, 85, p. 329-333), Subtilisin DY (Betzel et al. (1993), Arch. Biophys, 302, no. 2, p. 499-502), Lion Y (JP 04197182-A), RENNILASE® (Available from Novo Nordisk A/S), JA16 (WO 92/17576), ALCALASE® (a natural subtilisin Carlberg variant) (von der Osten et al., (1993), Journal of Biotechnology, 28, p. 55+).

[0109] Lipolytic Enzymes

[0110] Contemplated lipolytic enzymes include Humicola lanuginosalipases, e.g. the one described in EP 258 068 and EP 305 216 (See SEQ ID NO 6 below), Humicola insolens, a Rhizomucor mieheilipase, e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarcticalipase, e.g. the C. antarcticalipase A or B described in EP 214 761, a Pseudomonas lipasesuch as a P. alcaligenesand P. pseudoalcaligeneslipase, e.g. as described in EP 218 272, a P. cepacialipase, e.g. as described in EP 331 376, a Pseudomonas sp. lipase as disclosed in WO 95/14783, a Bacillus lipase, e.g. a B. subtilislipase (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260), a B. stearothermophiluslipase (JP 64/744992) and a B. pumiluslipase (WO 91/16422). Other types of lipolytic enzymes include cutinases, e.g. derived from Pseudomonas mendocinaas described in WO 88/09367, or a cutinase derived from Fusarium solanipisi (e.g. described in WO 90/09446).

[0111] Laccases

[0112] Contemplated laccases include Polyporus pinisituslaccase (WO 96/00290), Myceliophthora laccase(WO 95/33836) Scytalidium laccase(WO 95/338337), and Pyricularia oryzaelaccase (Available from Sigma).

[0113] Peroxidase

[0114] Contemplated peroxidases include B. pumilusperoxidases (WO 91/05858), Myxococcaceae peroxidase(WO 95/11964), Coprinus cinereus(WO 95/10602) and Arthromyces ramosusperoxidase (Kunishima et al. (1994), J. Mol. Biol. 235, p. 331-344).

[0115] Transglutaminases

[0116] Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S).

[0117] Protein Disulfide Isomerase

[0118] Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S).

[0119] The Polymeric Molecule

[0120] The polymeric molecules coupled to the polypeptide may be any suitable polymeric molecule, including natural and synthetic homo-polymers, such as polyols (i.e. poly-OH), polyamines (i.e. poly-NH₂) and polycarboxyl acids (i.e. poly—COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.

[0121] Examples of suitable polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched PEGS, poly-vinyl alcohol (PVA), poly-carboxylates, polyvinlypyrrlidone poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-starches and hydroxy propyl-starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenin, pectin, alginic acid hydrolysates and bio-polymers.

[0122] Preferred polymeric molecules are non-toxic polymeric molecules such as (m)polyethylene glycol ((m)PEG) which further requires a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface.

[0123] Generally seen polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG and especially mPEG, are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the like, have few reactive groups capable of cross-linking.

[0124] Even though all of the above mentioned polymeric molecules may be used according to the invention the methoxypolyethylene glycols (mPEG) may advantageously be used. This arises from the fact that methoxyethylene glycols have only one reactive end capable of conjugating with the enzyme. Consequently, the risk of cross-linking is less pronounced. Further, it makes the product more homogeneous and the reaction of the polymeric molecules with the enzyme easier to control.

[0125] Preparation of Enzyme Variants

[0126] Enzyme variants to be conjugated may be constructed by any suitable method. A number of methods are well established in the art. For instance enzyme variants according to the invention may be generated using the same materials and methods described in e.g. WO 89/06279 (Novo Nordisk A/S), EP 130,756 (Genentech), EP 479,870 (Novo Nordisk A/S), EP 214,435 (Henkel), WO 87/b4461 (Amgen), WO 87/05050 (Genex), EP application no. 87303761 (Genentech), EP 260,105 (Genencor), WO 88/06624 (Gist-Brocades NV), WO 88/07578 (Genentech), WO 20 88/08028 (Genex), WO 88/08033 (Amgen), WO 88/08164 (Genex), Thomas et al. (1985) Nature, 318 375-376; Thomas et al. (1987) J. Mol. Biol., 193, 803-813; Russel and Fersht (1987) Nature 328 496-500.

[0127] Generation of Site Directed Mutations

[0128] Prior to mutagenesis the gene encoding the polypeptide of interest must be cloned in a suitable vector. Methods for generating mutations in specific sites is described below.

[0129] Once the polypeptide encoding gene has been cloned, and desirable sites for mutation identified and the residue to substitute for the original ones have been decided, these mutations can be introduced using synthetic oligonucleotides.

[0130] These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligo-nucleotide synthesis. In a preferred method, Site-directed mutagenesis is carried out by SOE-PCR mutagenesis technique described by Kammann et al. (1989) Nucleic Acids Research 17(13), 5404, and by Sarkar G. and Sommer, S. S. (1990); Biotechniques 8, 404-407.

[0131] Activation of Polymers

[0132] If the polymeric molecules to be conjugated with the polypeptide in question are not active it must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the polymeric molecules to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.

[0133] Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), “Protein immobilization Fundamentals and applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.

[0134] In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.

[0135] Coupling polymeric molecules to the free acid groups of poly-peptides may be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).

[0136] Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.

[0137] Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.

[0138] Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.

[0139] Techniques involving coupling electrophilically activated PEGs to the amino groups of Lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

[0140] Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.

[0141] Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.

[0142] Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to Lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise.to carbamate linkages to the peptide.

[0143] Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.

[0144] Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imide thrones (U.S. Pat. No. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds are very high but may make the hydrolysis to fast.

[0145] PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.

[0146] Furthermore, a special linker can be introduced. The oldest being cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

[0147] Coupling of PEG to an aromatic amine followed by, diazotization yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.

[0148] As some peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.

[0149] PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95111924, Greenwald et al.). Lysine residues may also be used as the backbone.

[0150] The coupling technique used in the examples is, the N-succinimidyl carbonate conjugation technique, described in WO 90/13590 (Enzon).

[0151] Method for Preparing Improved Conjugates

[0152] It is also an object of the invention to provide a method for preparing improved polypeptide-polymer conjugates comprising the steps of:

[0153] a) identifying amino acid residues located on the surface of the 3D structure of the parent polypeptide in question,

[0154] b) selecting target amino acid residues on the surface of said 3D structure of said parent polypeptide to be mutated,

[0155] c) i) substituting or inserting one or more amino acid residues selected in step b) with an amino acid residue having a suitable attachment group, and/or ii) substituting or deleting one or more amino acid residues selected in step b) at or close to the functional site(s),

[0156] d) coupling polymeric molecules to the mutated polypeptide.

[0157] Step a) Identifyinq Amino Acid Residues Located on the Surface of the Parent Polypeptide

[0158] 3-dimensional Structure (3D-structure)

[0159] To perform the method of the invention a 3-dimensional structure of the parent polypeptide in question is required. This structure may for example be an X-ray structure, an NMR structure or a model-built structure. The Brookhaven Databank is a source of X-ray- and NMR-structures.

[0160] A model-built structure may be produced by the person skilled in the art if one or more 3D-structure(s) exist(s) of homologous polypeptide(s) sharing at least 30% sequence identity with the polypeptide in question. Several software packages exist which may be employed to construct a model structure. One example is the Homology 95.0 package from Biosym.

[0161] Typical actions required for the construction of a model structure are: alignment of homologous sequences for which 3D-structures exist, definition of Structurally Conserved Regions (SCRs), assignment of coordinates to SCRs, search for structural fragments/loops in structure databases to replace Variable Regions, assignment of coordinates to these regions, and structural refinement by energy minimization. Regions containing large inserts (≧3 residues) relative to the known 3D-structures are known to be quite difficult to model, and structural predictions must be considered with care.

[0162] Having obtained the 3D-structure of the polypeptide in question, or a model of the structure based on homology to known structures, this structure serves as an essential prerequisite for the fulfillment of the method described below.

[0163] Step b) Selection of Target Amino Acid Residues for Mutation

[0164] Target amino acid residues to be mutated are according to the invention selected in order to obtain additional or fewer attachment groups, such as free amino groups (—NH₂) or free carboxylic acid groups (—COOH), on the surface of the polypeptide and/or to obtain a more complete and broadly spread shielding of the epitope(s) on the surface of the polypeptide.

[0165] Conservative Substitution

[0166] It is preferred to make conservative substitutions in the polypeptide, as conservative substitutions secure that the impact of the mutation on the polypeptide structure is limited.

[0167] In the case of providing additional amino groups this may be done by substitution of Arginine to Lysine, both residues being positively charged, but only the Lysine having a free amino group suitable as an attachment group.

[0168] In the case of providing additional carboxylic acid groups the conservative substitution may for instance be an Aspargine to Aspartic acid or Glutamine to Glutamic acid substitution. These residues resemble each other in size and shape, except from the carboxylic groups being present on the acidic residues.

[0169] In the case of providing fewer attachment groups, e.g. at or close to the active site, a Lysine may be substituted with an Arginine, and so on.

[0170] Which amino acids to substitute depends in principle on the coupling chemistry to be applied.

[0171] Non-conservative Substitution

[0172] The mutation may also be on target amino acid residues which are less/non-conservative. Such mutation is suitable for obtaining a more complete and broadly spread shielding of the polypeptide surface than can be obtained by the conservative substitutions.

[0173] The method of the invention is first described in general terms, and subsequently using specific examples.

[0174] Note the use of the following terms:

[0175] Attachment_residue: residue(s) which can bind polymeric molecules, e.g. Lysines (amino group) or Aspartic/Glutamic acids (carboxylic groups). N- or C-terminal amino/carboxylic groups are to be included where relevant. Mutation_residue: residue(s) which is to be mutated, e.g. Arginine or Aspargine/Glutamine. Essential_catalytic_residues: residues which are known to be essential for catalytic function, e.g. the catalytic triad in Serine proteases. Solvent_exposed_residues: These are defined as residues which are at least 5% exposed according to the BIOSYM/INSIGHT algorithm found in the module Homology 95.0. The sequence of commands are as follows:

[0176] Homology=>ProStat=>Access_Surf=>Solv_Radius 1.4; Heavy atoms only; Radii source VdW; Output: Fractional Area; Polarity source: Default. The file filename_area.tab is produced. Note: For this program to function properly all water molecules must first be removed from the structure. It looks for example like:

[0177] 1. Identification of residues which are more than 10 Å away from the closest attachment_residue, and which are located at least 8 Å away from essential_catalytic_residues. This residue subset is called REST, and is the primary region for conservative mutation_residue to attachment_residue substitutions.

[0178] 2. Identification of residues which are located in a 0-5 Å shell around subset REST, but at least 8 Å away from essential_catalytic_residues. This residue subset is called SUB5B. This is a secondary region for conservative mutation_residue to attachment_residue substitutions, as a ligand bound to an attachment_residue in SUB5B will extend into the REST region and potentially prevent epitope recognition.

[0179] 3. Identification of solvent_exposed mutation_residues in REST and SUB5B as potential mutation sites for introduction of attachment_residues.

[0180] 4. Use BIOSYM/INSIGHT's Biopolymer module and replace residues identified under action 3.

[0181] 5. Repeat 1-2 above producing the subset RESTx. This subset includes residues which are more than 10 Å away from the nearest attachment_residue, and which are located at least 8 Å away from essential catalytic residues.

[0182] 6 Identify solvent_exposed_residues in RESTx. These are potential sites for less/non-conservative mutations to introduce attachment_residues.

[0183] Step c) Substituting, Inserting or Deleting Amino Acid Residues

[0184] The mutation(s) performed in step c) may be performed by standard techniques well known in the art, such as site-directed mutagenesis (see, e.g., Sambrook et al. (1989), Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, N.Y.

[0185] A general description of nucleotide substitution can be found in e.g. Ford et al., 1991, Protein Expression and Purification 2, p. 95-107.

[0186] Step d) Coupling Polymeric Molecules to the Modified Parent Enzyme

[0187] Polypeptide-polymer conjugates of the invention may be prepared by any coupling method known in the art including the above mentioned techniques.

[0188] Coupling of Polymeric Molecules to the Polypeptide in Question

[0189] If the polymeric molecules to be conjugated with the polypeptide are not active it must be activated by the use of a suitable method. The polymeric molecules may be coupled to the polypeptide through a linker. Suitable linkers are well known to the skilled person.

[0190] Methods and chemistry for activation of polymeric molecules as well as for conjugation of polypeptides are intensively described in the literature. Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde biepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine etc. (see R. F. Taylor, (1991), “Protein immobilization, Fundamentals and applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”, Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.

[0191] In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.

[0192] Coupling polymeric molecules to the free acid groups of enzymes can be performed with the aid of diimide and for example amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Amr. Chem. Soc., 98, 289-291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press).

[0193] Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.

[0194] Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho-pyridyl disulfide. Also vinylsulfone (U.S. Pat. No. 5,414,135, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.

[0195] Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.

[0196] Techniques involving coupling electrophilically activated PEGs to the amino groups of Lysines are also be useful. Many of the usual leaving groups for alcohols give rise to an amine linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135; Mosbach, K., Ed.; Academic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods in Enzymology vol. 135, Mosbach, K., Ed., Academic Press: Orlando, 1987; pp 79-84; Crossland et al., (1971), J. Amr. Chem. Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), supra; Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp. 341-352), aryl sulfonates like tosylates, and para-nitrobenzene sulfonates can be used.

[0197] Organic sulfonyl chlorides, e.g. Tresyl chloride, effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity), and satisfy the non-destructive requirements to the polypeptide.

[0198] Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.

[0199] Converting PEG into a chloroformate with phosgene gives rise to carbamate linkages to Lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (U.S. Pat. No. 5,122,614, (1992); Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.) etc. The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.

[0200] Furthermore, isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.

[0201] Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imide thrones (U.S. Pat. No. 5,349,001, (1994), Greenwald et al.). The reactivity of these compounds are very high but may make the hydrolysis to fast.

[0202] PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conjugate much more susceptible to hydrolysis (U.S. Pat. No. 5,122,614, (1992), Zalipsky). This group may be activated with N-hydroxy succinimide.

[0203] Furthermore, a special linker can be introduced. The oldest being cyanuric chloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat. No. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 375-378.

[0204] Coupling of PEG to an aromatic amine followed by diazotization yields a very reactive diazonium salt which in situ can be reacted with a peptide. An amide linkage may also be obtained by reacting an azlactone derivative of PEG (U.S. Pat. No. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.

[0205] As some peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1,3-diamino-2-propanol.

[0206] PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.

[0207] Specific Examples of PD498 Variant-SPEG Conjugates

[0208] A specific example of a protease is the parent PD498 (WO 93/24623 and SEQ ID NO. 2). The parent PD498 has a molecular weight of 29 kDa.

[0209] Lysine and Arginine Residues are Located as Follows:

[0210] The inventors examined which parent PD498 sites on the surface may be suitable for introducing additional attachment groups.

[0211] A. Suitable conservative Arginine to Lysine substitutions in parent PD498 may be any of R51K, R62K, R121K, R169K, R250K, R28K, R190K.

[0212] B. Suitable non-conservative substitutions in parent PD498 may be any of P6K, Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K, N65K, G87K, I88K, N209K, A211K, N216K, N217K, G218K, Y219K, S220K, Y221K, G262K.

[0213] As there is no Lysine residues at or close to the active site there is no need for removing any attachment group.

[0214] PD498 variant-SPEG conjugates may be prepared using any of the above mentioned PD498 variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme. A specific example is described below.

[0215] Specific Examples of BPN′ Variant-SPEG Conjugates

[0216] A specific example of a protease having an attachment group in the active site is BPN′ which has 11 attachment groups (plus an N-terminal amino group): BPN′ has a molecular weight of 28 kDa.

[0217] Lysine and Arginine Residues are Located as Follows:

[0218] The Lysine residue located within 0-5 Å of the active site can according to the invention advantageously be removed. Specifically this may be done by a K94R substitution.

[0219] BPN′ variant-SPEG conjugates may be prepared using the above mentioned BPN′ variant as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme.

[0220] Specific Example of SAVINASE®-SPEG Conjugate

[0221] As described in Example 2 parent SAVINASE® (von der Osten et al., (1993), Journal of Biotechnology, 28, p. 55+ and SEQ ID NO. 3) may according to the invention have added a number of amino attachment groups to the surface and removed an amino attachment group close to the active site.

[0222] Any of the following substitutions in the parent SAVINASE® are sites for mutagenesis: R10K, R19K, R45K, R145K, R170K, R186K and R247K.

[0223] The substitution K94R are identified as a mutation suitable for preventing attachment of polymers close to active site. SAVINASE® variant-SPEG conjugates may be prepared using any of the above mentioned SAVINASE® variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme.

[0224] A Specific Examples of Humicola LanuginosaLipase Variants-SPEG Conjugates

[0225] Specific examples of lipase variants with reduced immunogenicity using the parent Huminocal lanuginosaDSM 4109 lipase (see SEQ ID No 6) as the backbone for substitutions are listed below.

[0226] The parent unmodified Humicola lanuginosalipase has 8 attachment groups including the N-terminal NH₂group and a molecular weight of about 29 kDa. A. Suitable conservative Arginine to Lysine substitutions in the parent lipase may be any of R133K, R139K, R160K, R179K, R209K, R118K and R125K.

[0227] Suitable non-conservative substitutions in the parent lipase may be any of:

[0228] A18K,G31K,T32K,N33K,G38K,A40K,D48K,T50K,E56K,D57K,S58K,G59K, V60K, G61K,D62K,T64K,L78K,N88K,G91K,N92K,L93K,S105K,G106K, V120K,P136K,G225K,L227K,V228K,P229K,P250K,F262K.

[0229] Further suitable non-conservative substitution in the Humicola lanuginosalipase include: E87K or D254K.

[0230] Lipase variant-SPEG conjugates may be prepared using any of the above mentioned lipase variants as the starting material by any conjugation technique known in the art for coupling polymeric molecules to amino groups on the enzyme. A specific example is described below.

[0231] In Example 12 below it is shown that a conjugate of the Humicola lanuginosalipase variant with a E87K+D254K substitutions coupled to S-PEG 15,000 has reduced immunogenic response in Balb/C mice in comparison to the corresponding parent unmodified enzyme.

[0232] Immunogenicity and Allergenicity

[0233] “Immunogenicity” is a broader term than “antigenicity” and “allergenicity”, and expresses the immune system's response to the presence of foreign substances. Said foreign substances are called immunogens, antigens and allergens depending of the type of immune response they elicit.

[0234] An “immunogen” may be defined as a substance which, when introduced into circulatory system of animals and humans, is capable of stimulating an immunologic response resulting in formation of immunoglobulin.

[0235] The term “antigen” refers to substances which by themselves are capable of generating antibodies when recognized as a non-self molecule.

[0236] Further, an “allergen” may be defined as an antigen which may give rise to allergic sensitization or an allergic response by IgE antibodies (in humans, and molecules with comparable effects in animals).

[0237] Assessment of Immunogencity

[0238] Assessment of the immunogenicity may be made by injecting animal subcutaneously to enter the immunogen into the circulation system and comparing the response with the response of the corresponding parent polypeptide.

[0239] The “circulatory system” of the body of humans and animals means, in the context of the present invention, the system which mainly consists of the heart and blood vessels. The heart delivers the necessary energy for maintaining blood circulation in the vascular system. The circulation system functions as the organism's transportation system, when the blood transports O₂, nutritious matter, hormones, and other substances of importance for the cell regulation into the tissue. Further the blood removes CO₂from the tissue to the lungs and residual substances to e.g. the kidneys. Furthermore, the blood is of importance for the temperature regulation and the defense mechanisms of the body, which include the immune system.

[0240] A number of in vitro animal models exist for assessment of the immunogenic potential of polypeptides. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a mice model.

[0241] This model, seeks to identify the immunogenic response in the form of the IgG response in Balb/C mice being injected subcutaneously with modified and unmodified polypeptides.

[0242] Also other animal models can be used for assessment of the immunogenic potential.

[0243] A polypeptide having “reduced immunogenicity” according to the invention indicates that the amount of produced antibodies, e.g. immunoglobulin in humans, and molecules with comparable effects in specific animals, which can lead to an immune response, is significantly decreased, when introduced into the circulatory system, in comparison to the corresponding parent polypeptide.

[0244] For Balb/C mice the IgG response gives a good indication of the immunigenic potential of polypeptides.

[0245] Assessment of Allergenicity

[0246] Assessment of allergenicity may be made by inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent enzymes with the corresponding modified enzymes according to the invention.

[0247] A number of in vivo animal models exist for assessment of the allegenicity of enzymes. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek to identify respiratory allergens as a function of elicitation reactions induced in previously sensitized animals. According to these models the alleged allergens are introduced intratracheally into the animals.

[0248] A suitable strain of guinea pigs, the Dunkin Hartley strain, do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgG1A and IgG1B (see e.g. Prentø, ATLA, 19, p. 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgG1A and IgG1B is a measure of the allergenicity level.

[0249] The Balb/C mice strain is suitable for intratracheal exposure. Balb/C mice produce IgE as the allergic response. More details on assessing respiratory allergens in guinea pigs and mice is described by Kimber et al.,(1996), Fundamental and Applied Toxicology, 33, p. 1-10.

[0250] Other animals such as rats, rabbits etc. may also be used for comparable studies.

[0251] Composition

[0252] The invention relates to a composition comprising a polypeptide-polymer conjugate of the invention.

[0253] The composition may be a pharmaceutical or industrial composition.

[0254] The composition may further comprise other polypeptides, proteins or enzymes and/or ingredients normally used in e.g. detergents, including soap bars, household articles, agrochemicals, personal care products, including skin care compositions, cleaning compositions for e.g. contact lenses, oral and dermal pharmaceuticals, composition use for treating textiles, compositions used for manufacturing food, e.g. baking, and feed etc.

[0255] Use of the Polypeptide-polymer Conjugate

[0256] The invention also relates to the use of the method of the invention for reducing the immune response of polypeptides.

[0257] It is also an object of the invention to use the polypeptide-polymer conjugate of the invention to reduce the allergenicity of industrial products, such as detergents, such as laundry, dish wash and hard surface cleaning detergents, and food or feed products.

[0258] Enzymes:

[0259] PD498: Protease of subtilisin type shown in WO 93/24623. The sequence of PD498 is shown in SEQ ID NO. 1 and 2. SAVINASE® (Available from Novo Nordisk A/S) Humicola lanuginosalipase: Available from Novo Nordisk as Lipolase® and is further described in EP 305,216. The DNA and protein sequence is shown in SEQ ID NO 5 and 6, respectively.

[0260] Strains:

[0261] B. subtilis309 and 147 are variants of Bacillus lentus, deposited with the NCIB and accorded the accession numbers NCIB 10309 and 10147, and described in U.S. Pat. No. 3,723,250 incorporated by reference herein.

[0262] E. coliMC 1000 (M. J. Casadaban and S. N. Cohen (1980); J. Mol. Biol. 138 179-207), was made r⁻, m⁺ by conventional methods and is also described in U.S. patent application Ser. No. 039,298.

[0263] Vectors:

[0264] pPD498: E. coli—B. subtilisshuttle vector (described in U.S. Pat. No. 5,621,089 under section 6.2.1.6) containing the wild-type gene encoding for PD498 protease (SEQ ID NO. 2). The same vector is used for mutagenesis in E. colias well as for expression in B. subtilis.

[0265] General Molecular Biology Methods:

[0266] Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990). Enzymes for DNA manipulations were used according to the specifications of the suppliers.

[0267] Materials, Chemicals and Solutions:

[0268] Horse Radish Peroxidase labeled anti-rat-Ig (Dako, DK, P162, # 031; dilution 1:1000).

[0269] Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).

[0270] Rat anti-mouse IgE (Serotec MCA419; dilution 1:100).

[0271] Biotin-labeled mouse anti-rat IgG1 monoclonal antibody (Zymed 03-9140; dilution 1:1000)

[0272] Biotin-labeled rat anti-mouse IgG1 monoclonal antibody (Serotec MCA336B; dilution 1:1000)

[0273] Streptavidin-horse radish peroxidase (Kirkeg{dot over (a)}rd & Perry 14-30-00; dilution 1:1000).

[0274] CovaLink NH₂plates (Nunc, Cat# 459439)

[0275] Cyanuric chloride (Aldrich)

[0276] Acetone (Merck)

[0277] Rat anti-Mouse IgG1, biotin (SeroTec, Cat# MCA336B)

[0278] Streptavidin, peroxidase (KPL)

[0279] Ortho-Phenylene-diamine (OPD) (Kem-en-Tec)

[0280] H₂O₂, 30% (Merck)

[0281] Tween 20 (Merck)

[0282] Skim Milk powder (Difco)

[0283] H₂SO₄(Merck)

[0284] Buffers and Solutions:

[0285] Washing buffer PBS, 0.05% (v/v) Tween 20

[0286] Blocking buffer PBS, 2% (wt/v) Skim Milk powder

[0287] Dilution buffer PBS, 0.05% (v/v) Tween 20, 0.5% (wt/v) Skim Milk powder

[0288] Citrate buffer (0.1M, pH 5.0-5.2 (1 liter))NaCitrate 20.60 g Citric acid 6.30 g

[0289] Activation of CovaLink Plates:

[0290] Make a fresh stock solution of 10 mg cyanuric chloride per ml acetone.

[0291] Just before use, dilute the cyanuric chloride stock solution into PBS, while stirring, to a final concentration of 1 mg/ml.

[0292] Add 100 ml of the dilution to each well of the CovaLink NH₂plates, and incubate for 5 minutes at room temperature.

[0293] Wash 3 times with PBS.

[0294] Dry the freshly prepared activated plates at 50° C. for 30 minutes.

[0295] Immediately seal each plate with sealing tape.

[0296] Preactivated plates can be stored at room temperature for 3 weeks when kept in a plastic bag.

[0297] Sodium Borate, borax (Sigma)

[0298] 3,3-Dimethyl glutaric acid (Sigma)

[0299] CaCl₂(Sigma)

[0300] Tresyl chloride (2,2,2-triflouroethansulfonyl chloride) (Fluka)

[0301] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Fluka)

[0302] N-Hydroxy succinimide (Fluka art. 56480))

[0303] Phosgene (Fluka art. 79380)

[0304] Lactose (Merck 7656)

[0305] PMSF (phenyl methyl sulfonyl flouride) from Sigma Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.

[0306] Colouring Substrate:

[0307] OPD: o-phenylene-diamine, (Kementec cat no. 4260)

[0308] Test Animals:

[0309] Dunkin Hartley guinea pigs (from Charles River, Del.)

[0310] Female Balb/C mice (about 20 grams) purchased from Bomholdtgaard, Ry, Denmark.

[0311] Equipment:

[0312] XCEL II (Novex)

[0313] ELISA reader (UVmax, Molecular Devices)

[0314] HPLC (Waters)

[0315] PFLC (Pharmacia)

[0316] Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW.

[0317] SLT: Fotometer from SLT LabInstruments

[0318] Size-exclusion chromatograph (Spherogel TSK-G2000 SW).

[0319] Size-exclusion chromatograph (Superdex 200, Pharmacia, SW) Amicon Cell

[0320] Enzymes for DNA Manipulations

[0321] Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restriction endonucleases, ligases etc., are obtained from New England Biolabs. Inc.

[0322] ELISA Procedure for Determination of IqG₁Positive Guinea Pigs

[0323] ELISA microtiter plates are coated with rabbit anti-PD498 1:8000 in carbonate buffer and incubated overnight at 4° C. The next day the plates are blocked with 2% BSA for 1 hour and washes 3 times with PBS Tween 20. 1 μg/ml PD498 is added to the plates and incubated for 1 hour, then washed 3 times with PBS Tween 20.

[0324] All guinea pig sera samples and controls are applied to the ELISA plates with 2 μl sera and 98 μl PBS, incubated for 1 hour and washed 3 times with PBS Tween 20.

[0325] Then goat anti-guinea pig IgG₁(1:4000 in PBS buffer (Nordic Immunology 44-682)) is applied to the plates, incubated for 1 hour and washed with PBS tween 20.

[0326] Alkaline phosphatase marked rabbit anti-goat 1:8000 (Sigma A4187) is applied and incubated for 1 hour, washed 2 times in PBS Tween20 and 1 time with diethanol amine buffer.

[0327] The marked alkaline phosphatase is developed using p-nitrophenyl phosphate for 30 minutes at 37° C. or until appropriate colour has developed.

[0328] The reaction is stopped using Stop medium (K₂HPO₄/HaH₃buffer comprising EDTA (pH 10)) and read at OD 405/650 using an ELISA reader.

[0329] Double blinds are included on all ELISA plates.

[0330] Positive and negative sera values are calculated as the average blind values added 2 times the standard deviation. This gives an accuracy of 95%.

[0331] Determination of the Molecule Weight

[0332] Electrophoretic separation of proteins was performed by standard methods using 4-20% gradient SDS poly acrylamide gels (Novex). Proteins were detected by silver staining. The molecule weight was measured relative to the mobility of Mark-12® wide range molecule weight standards from Novex.

[0333] Protease Activity

[0334] Analysis With Suc-Ala-Ala-Pro-Phe-pNa:

[0335] Proteases cleave the bond between the peptide and p-nitroaniline to give a visible yellow colour absorbing at 405 nm.

[0336] Buffer: e.g. Britton and Robinson buffer pH 8.3 Substrate: 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO). 100 μl of this is diluted into 10 ml with Britton and Robinson buffer.

[0337] The substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and ABS₄₀₅nm/min. The temperature should be controlled (20-50° C. depending on protease). This is a measure of the protease activity in the sample.

[0338] Proteolytic Activity

[0339] In the context of this invention proteolytic activity is expressed in Kilo NOVO Protease Units (KNPU). The activity is determined relatively to an enzyme standard (SAVINASE_), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50° C., pH 8.3, 9 min. reaction time, 3 min. measuring time. A folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.

[0340] A GU is a Glycine Unit, defined as the proteolytic enzyme activity which, under standard conditions, during a 15-minutes' incubation at 40° C., with N-acetyl casein as substrate, produces an amount of NH₂-group equivalent to 1 mmole of glycine.

[0341] Enzyme activity can also be measured using the PNA assay, according to reaction with the soluble substrate succinyl-alanine-alanine-proline-phenyl-alanine-para-nitrophenol, which is described in the Journal of American Oil Chemists Society, Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., and Smith, L. A., (1988).

[0342] Fermentation of PD498 Variants

[0343] Fermentation of PD498 variants in B. subtilisare performed at 30° C. on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml BPX medium for 5 days. In order to make an e.g. 2 liter broth 20 Erlenmeyer flasks are fermented simultaneously.

[0344] BPX: Composition (per liter)

[0345] The starch in the medium is liquefied with α-amylase and the medium is sterilized by heating at 120° C. for 45 minutes. After sterilization the pH of the medium is adjusted to 9 by addition of NaHCO₃to 0.1 M.

[0346] Purification of PD498 Variants

[0347] Approximately 1.6 litres of PD498 variant fermentation broth are centrifuged at 5000 rpm for 35 minutes in 1 litre beakers. The supernatants are adjusted to pH 7.0 using 10% acetic acid and filtered on Seitz Supra S100 filter plates. The filtrates are concentrated-to approximately 400 ml using an Amicon CH2A UF unit equipped with an Amicon S1Y10 UF cartridge. The UF concentrate is centrifuged and filtered prior to absorption at room temperature on a Bacitracin affinity column at pH 7. The PD498 variant is eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.0 dimethylgutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7.

[0348] The fractions with protease activity from the Bacitracin purification step are combined and applied to a 750 ml Sephadex G25 column (5 cm diameter) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 6.0. Fractions with proteolytic activity from the Sephadex G25 column are combined and applied to a 150 ml CM Sepharose CL 6B cation exchange column (5 cm diameter) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.1 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.0. The protease is eluted using a linear gradient of 0-0.5 M sodium chloride in 1 litres of the same buffer. Protease containing fractions from the CM Sepharose column are combined and filtered through a 2 μfilter.

[0349] Balb/C Mice IgG ELISA Procedure:

[0350] The antigen is diluted to 1 mg/ml in carbonate buffer.

[0351] 100 ml is added to each well.

[0352] The plates are coated overnight at 4° C.

[0353] Unspecific adsorption is blocked by incubating each well for 1 hour at room temperature with 200 ml blocking buffer.

[0354] The plates are washed 3× with 300 ml washing buffer.

[0355] Unknown mouse sera are diluted in dilution buffer, typically 10×, 20× and 40×, or higher.

[0356] 100 ml is added to each well.

[0357] Incubation is for 1 hour at room temperature.

[0358] Unbound material is removed by washing 3× with washing buffer.

[0359] The anti-Mouse IgG1 antibody is diluted 2000× in dilution buffer.

[0360] 100 ml is added to each well.

[0361] Incubation is for 1 hour at room temperature.

[0362] Unbound material is removed by washing 3× with washing buffer.

[0363] Streptavidine is diluted 1000× in dilution buffer.

[0364] 100 ml is added to each well.

[0365] Incubation is for 1 hour at room temperature.

[0366] Unbound material is removed by washing 3× with 300 ml washing buffer.

[0367] OPD (0.6 mg/ml) and H₂O₂(0.4 ml/ml) is dissolved in citrate buffer.

[0368] 100 ml is added to each well.

[0369] Incubation is for 10 minutes at room temperature.

[0370] The reaction is stopped by adding 100 ml H₂SO₄.

[0371] The plates are read at 492 nm with 620 nm as reference.

[0372] Immunisation of Mice

[0373] Balb C mice (20 grams) are immunized 10 times (intervals of 14 days) by subcutaneous injection of the modified or unmodified polypeptide in question, respectively by standard, procedures known in art.

[0374] Suitable Substitutions in PD498 for Addition of Amino Attachment Groups (—NH₂)

[0375] The 3D structure of parent PD498 was modeled as described above based on 59% sequence identity with Thermitase® (2tec.pdb).

[0376] The sequence of PD498 is (see SEQ ID NO. 2). PD498 residue numbering is used, 1-280.

[0377] The commands performed in Insight (BIOSYM) are shown in the command files makeKzone.bcl and makeKzone2.bcl below:

[0378] Conservative Substitutions:

[0379] makekzone.bcl

[0380] 1 Delete Subset *

[0381] 2 Color Molecule Atoms * Specified Specification 55,0,255

[0382] 3 Zone Subset LYS :lys:NZ Static monomer/residue 10 Color_Subset 255,255,0

[0383] 4 Zone Subset NTERM :1: N Static monomer/residue 10 Color_Subset 255,255,0

[0384] 5 #NOTE: editnextline ACTSITE residues according to the protein

[0385] 6 Zone Subset ACTSITE :39,72,226 Static monomer/residue 8 Color_Subset 255,255,0

[0386] 7 Combine Subset ALLZONE Union LYS NTERM

[0387] 8 Combine Subset ALLZONE Union ALLZONE ACTSITE

[0388] 9 #NOTE: editnextline object name according to the protein

[0389] 10 Combine Subset REST Difference PD498FINALMODEL ALLZONE

[0390] 11 List Subset REST Atom Output_File restatom.list

[0391] 12 List Subset REST monomer/residue Output_File restmole.list

[0392] 13 Color Molecule Atoms ACTSITE Specified Specification 255,0,0

[0393] 14 List Subset ACTSITE Atom Output_File actsiteatom.list

[0394] 15 List Subset ACTSITE monomer/residue Output_File actsitemole.list

[0395] 16 #

[0396] 17 Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset

[0397] 18 Combine Subset SUB5A Difference REST5A ACTSITE

[0398] 19 Combine Subset SUB5B Difference SUB5A REST

[0399] 20 Color Molecule Atoms SUB5B Specified Specification 255,255,255

[0400] 21 List Subset SUB5B Atom Output_File sub5batom.list

[0401] 22 List Subset SUB5B monomer/residue Output_File sub5bmole.list

[0402] 23 #Now identify sites for lys→arg substitutions and continue with makezone2.bcl

[0403] 24 #Use grep command to identify ARG in restatom.list, sub5batom.list & accsiteatom.list

[0404] Comments:

[0405] Lines 1-8: The subset ALLZONE is defined as those residues which are either within 10 Å of the free amino groups on lysines or the N-terminal, or within 8 Å of the catalytic triad residues 39, 72 and 226.

[0406] Line 10: The subset REST is defined as those residues not included in ALLZONE.

[0407] Lines 17-20: Subset SUB5B is defined as those residues in a 5 Å shell around REST, excluding residues within 8 Å of the catalytic residues.

[0408] Line 23-24: REST contains Arg62 and Arg169, SUB5B contains Arg51, Arg121, and Arg250. ACTSITE contains Arg103, but position 103 is within 8 Å from essential_catalytic residues, and thus not relevant.

[0409] The colour codes are: (255,0,255)=magenta, (255,255,0)yellow, (255,0,0) red, and (255, 255, 255)=white.

[0410] The substitutions R51K, R62K, R121K, R169K and R250K are identified in parent PD498 as suitable sites for mutagenesis. The residues are substituted below in section 2, and further analysis done:

[0411] Non-conservative Substitutions:

[0412] makeKzone2.bcl

[0413] 1 #sourcefile makezone2.bcl Claus von der Osten 961128

[0414] 2 #

[0415] 3 #having scanned lists (grep arg command) and identified sites for lys→arg substitutions

[0416] 4 #NOTE: editnextline object name according to protein

[0417] 5 Copy Object -To_Clipboard -Displace PD498FINALMODEL newmodel

[0418] 6 Biopolymer

[0419] 7 #NOTE: editnextline object name according to protein

[0420] 8 Blank Object On PD498FINALMODEL

[0421] 9 #NOTE: editnextlines with lys→arg positions

[0422] 10 Replace Residue newmodel:51 lys L

[0423] 11 Replace Residue newmodel:62 lys L

[0424] 12 Replace Residue newmodel:121 lys L

[0425] 13 Replace Residue newmodel:169 lys L

[0426] 14 Replace Residue newmodel:250 lys L

[0427] 15 #

[0428] 16 #Now repeat analysis done prior to arg→lys, now including introduced lysines

[0429] 17 Color Molecule Atoms newmodel Specified Specification 255,0,255

[0430] 18 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10 Color_Subset 255,255,0

[0431] 19 Zone Subset NTERMx newmodel:1:N Static monomer/residue 10 Color_Subset 255,255,0

[0432] 20 #NOTE: editnextline ACTSITEx residues according to the protein

[0433] 21 Zone Subset ACTSITEx newmodel:39,72,226 Static monomer/residue 8 Color_Subset 255,255,0

[0434] 22 Combine Subset ALLZONEx Union LYSx NTERMx

[0435] 23 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx

[0436] 24 Combine Subset RESTx Difference newmodel ALLZONEx

[0437] 25 List Subset RESTx Atom Output_File restatom.list

[0438] 26 List Subset RESTx monomer/residue Output_File restxmole.list

[0439] 27 #

[0440] 28 Color Molecule Atoms ACTSITEx Specified Specification 255,0,0

[0441] 29 List Subset ACTSITEx Atom Output_File actsitexatom.list

[0442] 30 List Subset ACTSITEx monomer/residue Output_File actsitexmole.list

[0443] 31 #

[0444] 32 #read restxatom.list or restxmole.list to identify sites for (not_arg)→lys subst. if needed

[0445] Comments:

[0446] Lines 1-15: Solvent exposed arginines in subsets REST and SUB5B are replaced by lysines. Solvent accessibilities are recalculated following arginine replacement.

[0447] Lines 16-23: The subset ALLZONEx is defined as those residues which are either within 10 Å of the free amino groups on Lysines (after replacement) or the N-terminal, or within 8 Å of the catalytic triad residues 39, 72 and 226.

[0448] Line 24-26: The subset RESTx is defined as those residues not included in ALLZONEx, i.e. residues which are still potential epitope contributors. of the residues in RESTx, the following are >5% exposed (see lists below): 6-7,9-12,43-45,65,87-88,209,211,216-221,262.

[0449] The following mutations are proposed in parent PD498: P6K, Y7K, S9K, A10K, Y11K, Q12K, D43K, Y44K, N45K, N65K, G87K, I88K, N209K, A211K, N216K, N217K, G218K, Y219K, S220K, Y221K, G262K. Relevant data for Example 1: Solvent accessibility data for PD498MODEL:

[0450] Subset REST:

[0451] restmole.list

[0452] Subset REST:

[0453] PD498FINALMODEL:6-7,9-12,43-46,61-63,65,87-89,111-114,117-118,131,

[0454] PD498FINALMODEL:137-139,158-159,169-171,173-174,180-181,209,211,

[0455] PD498FINALMODEL:216-221,232-233,262, E282H

[0456] restatom.list

[0457] Subset REST:

[0458] PD498FINALMODEL:PRO 6:N,CA,CD,C,O,CB,CG

[0459] PD498FINALMODEL:TYR 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0460] PD498FINALMODEL:SER 9:N,CA,C,O,CB,OG

[0461] PD498FINALMODEL:ALA 10:N,CA,C,O,CB

[0462] PD498FINALMODEL:TYR 11:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0463] PD498FINALMODEL:GLN 12:N,CA,C,O,CB,CG,CD,OE1,NE2

[0464] PD498FINALMODEL:ASP 43:N,CA,C,O,CB,CG,OD1,OD2

[0465] PD498FINALMODEL:TYR

[0466] 44:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0467] PD498FINALMODEL:ASN 45:N,CA,C,O,CB,CG,OD1,ND2

[0468] PD498FINALMODEL:HIS

[0469] 46:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[0470] PD498FINALMODEL:ASP 61:N,CA,C,O,CB,CG,OD1,OD2

[0471] PD498FINALMODEL:ARG

[0472] 62:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[0473] PD498FINALMODEL:ASP 63:N,CA,C,O,CB,CG,OD1,OD2

[0474] PD498FINALMODEL:ASN 65:N,CA,C,O,CB,CG,OD1,ND2

[0475] PD498FINALMODEL:GLY 87:N,CA,C,O

[0476] PD498FINALMODEL:ILE 88:N,CA,C,O,CB,CG1,CG2,CD1

[0477] PD498FINALMODEL:GLY 89:N,CA,C,O

[0478] PD498FINALMODEL:GLY 111:N,CA,C,O

[0479] PD498FINALMODEL:SER 112:N,CA,C,O,CB,OG

[0480] PD498FINALMODEL:LEU 113:N,CA,C,O,CB,CG,CD1,CD2

[0481] PD498FINALMODEL:ASP 114:N,CA,C,O,CB,CG,OD1,OD2

[0482] PD498FINALMODEL:ALA 117:N,CA,C,O,CB

[0483] PD498FINALMODEL:SER 118:N,CA,C,O,CB,OG

[0484] PD498FINALMODEL:LEU 131:N,CA,C,O,CB,CG,CD1,CD2

[0485] PD498FINALMODEL:CYS 137:N,CA,C,O,CB,SG

[0486] PD498FINALMODEL:GLU

[0487] 138:N,CA,C,O,CB,CG,CD,OE1,OE2

[0488] PD498FINALMODEL:CYS 139:N,CA,C,O,CB,SG

[0489] PD498FINALMODEL:VAL 158:N,CA,C,O,CB,CG1,CG2

[0490] PD498FINALMODEL:VAL 159:N,CA,C,O,CB,CG1,CG2

[0491] PD498FINALMODEL:ARG

[0492] 169:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[0493] PD498FINALMODEL:THR 170:N,CA,C,O,CB,OG1,CG2

[0494] PD498FINALMODEL:PHE

[0495] 171:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[0496] PD498FINALMODEL:PRO 173:N,CA,CD,C,O,CB,CG

[0497] PD498FINALMODEL:ALA 174:N,CA,C,O,CB

[0498] PD498FINALMODEL:ILE 180:N,CA,C,O,CB,CG1,CG2,CD1

[0499] PD498FINALMODEL:ALA 181:N,CA,C,O,CB

[0500] PD498FINALMODEL:ASN 209:N,CA,C,O,CB,CG,OD1,ND2

[0501] PD498FINALMODEL:ALA 211:N,CA,C,O,CB

[0502] PD498FINALMODEL:ASN 216:N,CA,C,O,CB,CG,OD1,ND2

[0503] PD498FINALMODEL:ASN 217:N,CA,C,O,CB,CG,OD1,ND2

[0504] PD498FINALMODEL:GLY 218:N,CA,C,O

[0505] PD498FINALMODEL:TYR

[0506] 219:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0507] PD498FINALMODEL:SER 220:N,CA,C,O,CB,OG

[0508] PD498FINALMODEL:TYR

[0509] 221:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0510] PD498FINALMODEL:VAL 232:N,CA,C,O,CB,CG1,CG2

[0511] PD498FINALMODEL:ALA 233:N,CA,C,O,CB

[0512] PD498FINALMODEL:GLY 262:N,CA,C,O

[0513] PD498FINALMODEL:CA E282H:CA

[0514] Subset SUB5B:

[0515] sub5bmole.list

[0516] Subset SUB5B:

[0517] PD498FINALMODEL:4-5,8,13-16,34-35,47-51,53,64,83,85-86,90-91,120-124,

[0518] PD498FINALMODEL:128-130,140-141,143-144,147-148,151-152,156-157,

[0519] PD498FINALMODEL:165,167-168,172,175-176,178-179,196,200-205,208,

[0520] PD498FINALMODEL:234-237,250,253-254,260-261,263-267,272,E281H,

[0521] PD498FINALMODEL:E283H

[0522] sub5batom.list

[0523] Subset SUB5B:

[0524] PD498FINALMODEL:ASN.4:N,CA,C,O,CB,CG,OD1,ND2

[0525] PD498FINALMODEL:ASP 5:N,CA,C,O,CB,CG,OD1,OD2

[0526] PD498FINALMODEL:TYR

[0527] 8:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0528] PD498FINALMODEL:TYR

[0529] 13:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0530] PD498FINALMODEL:GLY 14:N,CA,C,O

[0531] PD498FINALMODEL:PRO 15:N,CA,CD,C,O, CB,CG

[0532] PD498FINALMODEL:GLN 16:N,CA,C,O,CB,CG,CD,OE1,NE2

[0533] PD498FINALMODEL:THR 34:N,CA,C,O,CB,OG1,CG2

[0534] PD498FINALMODEL:VAL 35:N,CA,C,O,CB,CG1,CG2

[0535] PD498FINALMODEL:PRO 47:N,CA,CD,C,O,CB,CG

[0536] PD498FINALMODEL:ASP 48:N,CA,C,O,CB,CG,OD1,OD2

[0537] PD498FINALMODEL:LEU 49:N,CA,C,O,CB,CG,CD1,CD2

[0538] PD498FINALMODEL:ALA 50:N,CA,C,O,CB

[0539] PD498FINALMODEL:ARG

[0540] 51:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[0541] PD498FINALMODEL:VAL 53:N,CA,C,O,CB,CG1,CG2

[0542] PD498FINALMODEL:ASN 64:N,CA,C,O,CB,CG,OD1,ND2

[0543] PD498FINALMODEL:ASP 83:N,CA,C,O,CB,CG,OD1,OD2

[0544] PD498FINALMODEL:ASN 85:N,CA,C,O,CB,CG,OD1,ND2

[0545] PD498FINALMODEL:ASN 86:N,CA,C,O,CB,CG,OD1,ND2

[0546] PD498FINALMODEL:VAL 90:N,CA, C,O,CB,CG1,CG2

[0547] PD498FINALMODEL:ALA 91:N,CA,C,O,CB

[0548] PD498FINALMODEL:ILE 120:N,CA,C,O,CB,CG1,CG2,CD1

[0549] PD498FINALMODEL:ARG

[0550] 121:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[0551] PD498FINALMODEL:TYR

[0552] 122:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0553] PD498FINALMODEL:ALA 123:N,CA,C,O,CB

[0554] PD498FINALMODEL:ALA 124:N,CA,C,O,CB

[0555] PD498FINALMODEL:ALA 128:N,CA,C,O,CB

[0556] PD498FINALMODEL:LYS 129:N,CA,C,O,CB,CG,CD,CE,NZ

[0557] PD498FINALMODEL:VAL 130:N,CA,C,O,CB,CG1,CG2

[0558] PD498FINALMODEL:ASN 140:N,CA,C,O,CB,CG,OD1,ND2

[0559] PD498FINALMODEL:SER 141:N,CA,C,O,CB,OG

[0560] PD498FINALMODEL:THR 143:N,CA,C,O,CB,OG1,CG2

[0561] PD498FINALMODEL:LEU 144:N,CA,C,O,CB,CG,CD1,CD2

[0562] PD498FINALMODEL:ALA 147:N,CA,C,O,CB

[0563] PD498FINALMODEL:VAL 148:N,CA,C,O,CB,CG1,CG2

[0564] PD498FINALMODEL:ALA 151:N,CA,C,O,CB

[0565] PD498FINALMODEL:TRP

[0566] 52:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3, CZ2,CZ3,CH2

[0567] PD498FINALMODEL:ALA 156:N,CA,C,O,CB

[0568] PD498FINALMODEL:VAL 157:N,CA,C,O,CB,CG1,CG2

[0569] PD498FINALMODEL:ASP 165:N,CA,C,O,CB,CG,OD1,OD2

[0570] PD498FINALMODEL:VAL 167:N,CA,C,O,CB,CG1,CG2

[0571] PD498FINALMODEL:SER 168:N,CA,C,O,CB,OG

[0572] PD498FINALMODEL:GLN

[0573] 172:N,CA,C,O,CB,CG,CD,OE1,NE2

[0574] PD498FINALMODEL:SER 175:N,CA,C,O,CB,OG

[0575] PD498FINALMODEL:TYR

[0576] 176:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0577] PD498FINALMODEL:ASN 178:N,CA,C,O,CB,CG,OD1,ND2

[0578] PD498FINALMODEL:ALA 179:N,CA,C,O,CB

[0579] PD498FINALMODEL:ASN 196:N,CA,C,O,CB,CG,OD1,ND2

[0580] PD498FINALMODEL:TRP

[0581] 200:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3, CZ2,CZ3,CH2

[0582] PD498FINALMODEL:VAL 201:N,CA,C,O,CB,CG1,CG2

[0583] PD498FINALMODEL:ASP 202:N,CA,C,O,CB,CG,OD1,OD2

[0584] PD498FINALMODEL:VAL 203:N,CA,C,O,CB,CG1,CG2

[0585] PD498FINALMODEL:THR 204:N,CA,C,O,CB,OG1,CG2

[0586] PD498FINALMODEL:ALA 205:N,CA,C,O,CB

[0587] PD498FINALMODEL:VAL 208:N,CA,C,O,CB,CG1,CG2

[0588] PD498FINALMODEL:GLY 234:N,CA,C,O

[0589] PD498FINALMODEL:LEU 235:N,CA,C,O,CB,CG,CD1,CD2

[0590] PD498FINALMODEL:ALA 236:N,CA,C,O,CB

[0591] PD498FINALMODEL:ALA 237:N,CA,C,O,CB

[0592] PD498FINALMODEL:ARG

[0593] 250:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[0594] PD498FINALMODEL:ILE 253:N,CA,C,O,CB,CG1,CG2,CD1

[0595] PD498FINALMODEL:GLU

[0596] 254:N,CA,C,O,CB,CG,CD,OE1,OE2

[0597] PD498FINALMODEL:ILE 260:N,CA,C,O,CB,CG1,CG2,CD1

[0598] PD498FINALMODEL:SER 261:N,CA,C,O,CB,OG

[0599] PD498FINALMODEL:THR 263:N,CA,C,O,CB,OG1,CG2

[0600] PD498FINALMODEL:GLY 264:N,CA,C,O

[0601] PD498FINALMODEL:THR 265:N,CA,C,O,CB,OG1,CG2

[0602] PD498FINALMODEL:ASN 266:N,CA,C,O,CB,CG,OD1,ND2

[0603] PD498FINALMODEL:PHE

[0604] 267:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[0605] PD498FINALMODEL:ILE 272:N,CA,C,O,CB,CG1,CG2,CD1

[0606] PD498FINALMODEL:CA E281H:CA

[0607] PD498FINALMODEL:CA E283H:NA

[0608] Subset ACTSITE:

[0609] actsitemole.list

[0610] Subset ACTSITE:

[0611] PD498FINALMODEL:36-42,57-60,66-80,100-110,115-116,119,132-136,160-164,

[0612] PD498FINALMODEL:182-184,194,206-207,210,212-215,222-231

[0613] actsiteatom.list

[0614] Subset ACTSITE:

[0615] PD498FINALMODEL:ALA 36:N,CA,C,O,CB

[0616] PD498FINALMODEL:VAL 37:N,CA,C,O,CB,CG1,CG2

[0617] PD498FINALMODEL:LEU 38:N,CA,C,O,CB,CG,CD1,CD2

[0618] PD498FINALMODEL:ASP 39:N,CA,C,O,CB,CG,OD1,OD2

[0619] PD498FINALMODEL:SER 40:N,CA,C,O,CB,OG

[0620] PD498FINALMODEL:GLY 41:N,CA,C,O

[0621] PD498FINALMODEL:VAL 42:N,CA,C,O,CB,CG1,CG2

[0622] PD498FINALMODEL:TYR

[0623] 57:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0624] PD498FINALMODEL:ASP 58:N,CA,C,O,CB,CG,OD1,OD2

[0625] PD498FINALMODEL:PHE

[0626] 59:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[0627] PD498FINALMODEL:ILE 60:N,CA,C,O,CB,CG1,CG2,CD1

[0628] PD498FINALMODEL:PRO 66:N,CA,CD,C,O,CB,CG

[0629] PD498FINALMODEL:MET 67:N,CA,C,O,CB,CG,SD,CE

[0630] PD498FINALMODEL:ASP 68:N,CA,C,O,CB,CG,OD1,OD2

[0631] PD498FINALMODEL:LEU 69:N,CA,C,O,CB,CG,CD1,CD2

[0632] PD498FINALMODEL:ASN 70:N,CA,C,O,CB,CG,OD1,ND2

[0633] PD498FINALMODEL:GLY 71:N,CA,C,O

[0634] PD498FINALMODEL:HIS

[0635] 72:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[0636] PD498FINALMODEL:GLY 73:N,CA,C,O

[0637] PD498FINALMODEL:THR 74:N,CA,C,O,CB,OG1,CG2

[0638] PD498FINALMODEL:HIS

[0639] 75:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[0640] PD498FINALMODEL:VAL 76:N,CA,C,O,CB,CG1,CG2

[0641] PD498FINALMODEL:ALA 77:N,CA,C,O,CB

[0642] PD498FINALMODEL:GLY 78:N,CA,C,O

[0643] PD498FINALMODEL:THR 79:N,CA,C,O,CB,OG1,CG2

[0644] PD498FINALMODEL:VAL 80:N,CA,C,O,CB,CG1,CG2

[0645] PD498FINALMODEL:LEU 100:N,CA,C,O,CB,CG,CD1,CD2

[0646] PD498FINALMODEL:ALA 101:N,CA,C,O,CB

[0647] PD498FINALMODEL:VAL 102:N,CA,C,O,CB,CG1,CG2

[0648] PD498FINALMODEL:ARG

[0649] 103:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[0650] PD498FINALMODEL:VAL 104:N,CA,C,O,CB,CG1,CG2

[0651] PD498FINALMODEL:LEU 105:N,CA,C,O,CB,CG,CD1,CD2

[0652] PD498FINALMODEL:ASP 106:N,CA,C,O,CB,CG,OD1,OD2

[0653] PD498FINALMODEL:ALA 107:N,CA,C,O,CB

[0654] PD498FINALMODEL:ASN 108:N,CA,C,O,CB,CG,OD1,ND2

[0655] PD498FINALMODEL:GLY 109:N,CA,C,O

[0656] PD498FINALMODEL:SER 110:N,CA,C,O,CB,OG

[0657] PD498FINALMODEL:SER 115:N,CA,C,O,CB,OG

[0658] PD498FINALMODEL:ILE 116:N,CA,C,O,CB,CG1,CG2,CD1

[0659] PD498FINALMODEL:GLY 119:N,CA,C,O

[0660] PD498FINALMODEL:ASN 132:N,CA,C,O,CB,CG,OD1,ND2

[0661] PD498FINALMODEL:LEU 133:N,CA,C,O,CB,CG,CD1,CD2

[0662] PD498FINALMODEL:SER 134:N,CA,C,O,CB,OG

[0663] PD498FINALMODEL:LEU 135:N,CA,C,O,CB,CG,CD1,CD2

[0664] PD498FINALMODEL:GLY 136:N,CA,C,O

[0665] PD498FINALMODEL:ALA 160:N,CA,C,O,CB

[0666] PD498FINALMODEL:ALA 161:N,CA,C,O,CB

[0667] PD498FINALMODEL:ALA 162:N,CA,C,O,CB

[0668] PD498FINALMODEL:GLY 163:N,CA,C,O

[0669] PD498FINALMODEL:ASN 164:N,CA,C,O,CB,CG,OD1,ND2

[0670] PD498FINALMODEL:VAL 182:N,CA,C,O,CB,CG1,CG2

[0671] PD498FINALMODEL:GLY 183:N,CA,C,O

[0672] PD498FINALMODEL:ALA 184:N,CA,C,O,CB

[0673] PD498FINALMODEL:PHE

[0674] 194:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[0675] PD498FINALMODEL:PRO 206:N,CA,CD,C,O,CB,CG

[0676] PD498FINALMODEL:GLY 207:N,CA,C,O

[0677] PD498FINALMODEL:ILE 210:N,CA,C,O,CB,CG1,CG2,CD1

[0678] PD498FINALMODEL:SER 212:N,CA,C,O,CB,OG

[0679] PD498FINALMODEL:THR 213:N,CA,C,O,CB,OG1,CG2

[0680] PD498FINALMODEL:VAL 214:N,CA,C,O,CB,CG1,CG2

[0681] PD498FINALMODEL:PRO 215:N,CA,CD,C,O,CB,CG

[0682] PD498FINALMODEL:MET 222:N,CA,C,O,CB,CG,SD,CE

[0683] PD498FINALMODEL:SER 223:N,CA,C,O,CB,OG

[0684] PD498FINALMODEL:GLY 224:N,CA,C,O

[0685] PD498FINALMODEL:THR 225:N,CA,C,O,CB,OG1,CG2

[0686] PD498FINALMODEL:SER 226:N,CA,C,O,CB,OG

[0687] PD498FINALMODEL:MET 227:N,CA,C,O,CB,CG,SD,CE

[0688] PD498FINALMODEL:ALA 228:N,CA,C,O,CB

[0689] PD498FINALMODEL:SER 229:N,CA,C,O,CB,OG

[0690] PD498FINALMODEL:PRO 230:N,CA,CD,C,O,CB,CG

[0691] PD498FINALMODEL:HIS

[0692] 231:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[0693] Subset RESTx:

[0694] restxmole.list

[0695] Subset RESTx:

[0696] NEWMODEL:6-7,9-12,43-46,65,87-89,131,173,209,211,216-221,232-233,

[0697] NEWMODEL:262,E282H

[0698] restxatom.list

[0699] Subset RESTx:

[0700] NEWMODEL:PRO 6:N,CA,CD,C,O,CB,CG

[0701] NEWMODEL:TYR 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0702] NEWMODEL:SER 9:N,CA,C,O,CB,OG

[0703] NEWMODEL:ALA 10:N,CA,C,O,CB

[0704] NEWMODEL:TYR 11:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0705] NEWMODEL:GLN 12:N,CA,C,O,CB,CG,CD,OE1,NE2

[0706] NEWMODEL:ASP 43:N,CA,C,O,CB,CG,OD1,OD2

[0707] NEWMODEL:TYR 44:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0708] NEWMODEL:ASN 45:N,CA,C,O,CB,CG,OD1,ND2

[0709] NEWMODEL:HIS 46:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[0710] NEWMODEL:ASN 65:N,CA,C,O,CB,CG,OD1,ND2

[0711] NEWMODEL:GLY 87:N,CA,C,O

[0712] NEWMODEL:ILE 88:N,CA,C,O,CB,CG1,CG2,CD1

[0713] NEWMODEL:GLY 89:N,CA,C,O

[0714] NEWMODEL:LEU 131:N,CA,C,O,CB,CG,CD1,CD2

[0715] NEWMODEL:PRO 173:N,CA,CD,C,O,CB,CG

[0716] NEWMODEL:ASN 209:N,CA,C,O,CB,CG,OD1,ND2

[0717] NEWMODEL:ALA 211:N,CA,C,O,CB

[0718] NEWMODEL:ASN 216:N,CA,C,O,CB,CG,OD1,ND2

[0719] NEWMODEL:ASN 217:N,CA,C,O,CB,CG,OD1,ND2

[0720] NEWMODEL:GLY 218:N,CA,C,O

[0721] NEWMODEL:TYR 219:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0722] NEWMODEL:SER 220:N,CA,C,O,CB,OG

[0723] NEWMODEL:TYR 221:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[0724] NEWMODEL:VAL 232:N,CA,C,O,CB,CG1,CG2

[0725] NEWMODEL:ALA 233:N,CA,C,O,CB

[0726] NEWMODEL:GLY 262:N,CA,C,O

[0727] NEWMODEL:CA E282H:CA

[0728] Suitable Substitutions in SAVINASE® for Addition of Amino Attachment Groups (—NH₂)

[0729] The known X-ray structure of SAVINASE® was used to find where suitable amino attachment groups may is added (Betzel et al, (1992), J. Mol. Biol. 223,p 427-445).

[0730] The 3D structure of, SAVINASE® is available in the Brookhaven Databank as 1svn.pbd. A related subtilisin is available as 1st3.pdb.

[0731] The sequence of SAVINASE® is shown in SEQ ID NO. 3 The sequence numbering used is that of subtilisin BPN′, SAVINASE® having deletions relative to BPN′ at positions: 36, 56, 158-159 and 163-164. The active site residues (functional site) are D32,H64 and S221.

[0732] The commands performed in Insight (BIOSYM) are shown in the command files makeKzone.bcl and makeKzone2.bcl below:

[0733] Conservative Substitutions:

[0734] makekzone.bcl

[0735] Delete Subset *

[0736] Color Molecule Atoms * Specified Specification 255,0,255

[0737] Zone Subset LYS :lys:NZ Static monomer/residue 10 Color_Subset 255,255,0

[0738] Zone Subset NTERM :e1:N Static monomer/residue 10 Color_Subset 255,255,0

[0739] #NOTE: editnextline ACTSITE residues according to the protein

[0740] Zone Subset ACTSITE :e32,e64,e221 Static monomer/residue 8

[0741] Color_Subset 255,255,0

[0742] Combine Subset ALLZONE Union LYS NTERM

[0743] Combine Subset ALLZONE Union ALLZONE ACTSITE

[0744] #NOTE: editnextline object name according to the protein

[0745] Combine Subset REST Difference SAVI8 ALLZONE

[0746] List Subset REST Atom Output_File restatom.list

[0747] List Subset REST monomer/residue Output_File restmole.list

[0748] Color Molecule Atoms ACTSITE Specified Specification 255,0,0

[0749] List Subset ACTSITE Atom Output_File actsiteatom.list

[0750] List Subset ACTSITE monomer/residue Output_File

[0751] actsitemole.list

[0752] #

[0753] Zone Subset REST5A REST Static Monomer/Residue 5-Color_Subset

[0754] Combine Subset SUB5A Difference REST5A ACTSITE

[0755] Combine Subset SUB5B Difference SUB5A REST

[0756] Color Molecule Atoms SUB5B Specified Specification 255,255,255

[0757] List Subset SUB5B Atom Output_File sub5batom.list

[0758] List Subset SUB5B monomer/residue Output_File sub5bmole.list

[0759] #Now identify sites for lys→arg substitutions and continue with makezone2.bcl

[0760] #Use grep command to identify ARG in restatom.list,

[0761] sub5batom.list & accsiteatom.list

[0762] Comments: 4

[0763] In this case of SAVINASE® REST contains the Arginines Arg10, Arg170 and Arg 186, and SUB5B contains Arg19, Arg45, Arg145 and Arg247.

[0764] These residues are all solvent exposed. The substitutions R10K, R19K, R45K, R145K, R170K, R186K and R247K are identified in SAVINASE® as sites for mutagenesis within the scope of this invention. The residues are substituted below in section 2, and further analysis done. The subset ACTSITE contains Lys94.

[0765] The substitution K94R is a mutation removing Lysine as attachment group close to the active site.

[0766] Non-conservative Substitutions:

[0767] makeKzone2.bcl

[0768] #sourcefile makezone2.bcl Claus von der Osten 961128

[0769] #

[0770] #having scanned lists (grep arg command) and identified sites for lys→arg substitutions

[0771] #NOTE: editnextline object name according to protein

[0772] Copy Object -To_Clipboard -Displace SAVE8 newmodel Biopolymer

[0773] #NOTE: editnextline object name according to protein Blank Object On SAVI8

[0774] #NOTE: editnextlines with lys→arg positions

[0775] Replace Residue newmodel:e10 lys L

[0776] Replace Residue newmodel:e170 lys L

[0777] Replace Residue newmodel:e186 lys L

[0778] Replace Residue newmodel:e19 lys L

[0779] Replace Residue newmodel:e45 lys L

[0780] Replace Residue newmodel:e145 lys L

[0781] Replace Residue newmodel:e241 lys L

[0782] #Now repeat analysis done prior to arg→lys, now including

[0783] introduced lysines

[0784] Color Molecule Atoms newmodel Specified Specification 255,0,255

[0785] Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10

[0786] Color_Subset 255,255,0

[0787] Zone Subset NTERMx newmodel:e1:N Static monomer/residue 10

[0788] Color_Subset 255,255,0

[0789] #NOTE: editnextline ACTSITEx residues according to the protein

[0790] Zone Subset ACTSITEx newmodel:e32,e64,e221 Static

[0791] monomer/residue 8 Color_Subset 255,255,0

[0792] Combine Subset ALLZONEx Union LYSx NTERMx

[0793] Combine Subset ALLZONEx Union ALLZONEx ACTSITEx

[0794] Combine Subset RESTx Difference newmodel ALLZONEx

[0795] List Subset RESTx Atom Output_File restxatom.list

[0796] List Subset RESTx monomer/residue Output_File restxmole.list

[0797] Color Molecule Atoms ACTSITEx Specified Specification 255,0,0

[0798] List Subset ACTSITEx Atom Output_File actsitexatom.list

[0799] List Subset ACTSITEx monomer/residue Output_File

[0800] actsitexmole.list

[0801] #

[0802] #read restxatom.list or restxmole.list to identify sites for (not_arg)→lys subst. if needed

[0803] Comments:

[0804] Of the residues in RESTx, the following are >5% exposed (see lists below): 5,14,22,38-40,42,75-76,82,86,103-105,108,133-135,137,140,173,204,206,211-213,215-216,269. The following mutations are proposed in SAVINASE®: P5K, P14K, T22K, T38K, H39K, P40K, L42K, L75K, N76K, L82K, P86K, S103K, V104K, S105K, A108K, A133K, T134K, L135K, Q137K, N140K, N173K, N204K, Q206K, G211K, S212K, T213K, A215K, S216K, N269K. Relevant data for Example 2:

[0805] Solvent Accessibility Data for SAVINASE®:

[0806] Subset REST:

[0807] restmole.list

[0808] Subset REST:

[0809] SAVI8:E5-E15,E17-E18,E22,E38-E40,E42-E43,E73-E76,E82-E86,E103-E105,

[0810] SAVI8:E108-E109,E111-E112,E115-E116,E122,E128-E144,E149-E150,E156-E157,

[0811] SAVI8:E160-E162,E165-E168,E170-E171,E173,E180-E188,E190-E192,E200,

[0812] SAVI8:E203-E204,E206,E211-E213,E215-E216,E227-E230,E255-E259,E261-E262,

[0813] SAVI8:E267-E269

[0814] restatom.list

[0815] Subset REST:

[0816] SAVI8:PRO E5:N,CD,CA,CG,CB,C,O

[0817] SAVI8:TRP E6:N,CA,CD2,CE2,NE1,CD1,CG,CE3,CZ3,CH2,CZ2,CB,C,O

[0818] SAVI8:GLY E7:N,CA,C,O

[0819] SAVI8:ILE E8:N,CA,CD1,CG1,CB,CG2,C,O

[0820] SAVI8:SER E9:N,CA,OG,CB,C,O

[0821] SAVI8:ARG E10:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0822] SAVI8:VAL E11:N,CA,CG2,CG1,CB,C,O

[0823] SAVI8:GLN E12:N,CA,NE2,OE1,CD,CG,CB,C,O

[0824] SAVI8:ALA E13:N,CA,CB,C,O

[0825] SAVI8:PRO E14:N,CD,CA,CG,CB,C,O

[0826] SAVI8:ALA E15:N,CA,CB,C,O

[0827] SAVI8:HIS E17:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[0828] SAVI8:ASN E18:N,CA,ND2,OD1,CG,CB,C,O

[0829] SAVI8:THR E22:N,CA,CG2,OG1,CB,C,O

[0830] SAVI8:THR E38:N,CA,CG2,OG1,CB,C,O

[0831] SAVI8:HIS E39:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[0832] SAVI8:PRO E40:N,CD,CA,CG,CB,C,O

[0833] SAVI8:LEU E42:N,CA,CD2,CD1,CG,CB,C,O

[0834] SAVI8:ASN E43:N,CA,ND2,OD1,CG,CB,C,O

[0835] SAVI8:ALA E73:N,CA,CB,C,O

[0836] SAVI8:ALA E74:N,CA,CB,C,O

[0837] SAVI8:LEU E75:N,CA,CD2,CD1,CG,CB,C,O

[0838] SAVI8:ASN E76:N,CA,ND2,OD1,CG,CB,C,O

[0839] SAVI8:LEU E82:N,CA,CD2,CD1,CG,CB,C,O

[0840] SAVI8:GLY E83:N,CA,C,O

[0841] SAVI8:VAL E84:N,CA,CG2,CG1,CB,C,O

[0842] SAVI8:ALA E85:N,CA,CB,C,O

[0843] SAVI8:PRO E86:N,CD,CA,CG,CB,C,O

[0844] SAVI8:SER E103:N,CA,OG,CB,C,O

[0845] SAVI8:VAL E104:N,CA,CG2,CG1,CB,C,O

[0846] SAVI8:SER E105:N,CA,OG,CB,C,O

[0847] SAVI8:ALA E108:N,CA,CB,C,O

[0848] SAVI8:GLN E109:N,CA,NE2,OE1,CD,CG,CB,C,O

[0849] SAVI8:LEU E111:N,CA,CD2,CD1,CG,CB,C,O

[0850] SAVI8:GLU E112:N,CA,OE2,OE1,CD,CG,CB,C,O

[0851] SAVI8:GLY E115:N,CA,C,O

[0852] SAVI8:ASN E116:N,CA,ND2,OD1,CG,CB,C,O

[0853] SAVI8:ALA E122:N,CA,CB,C,O

[0854] SAVI8:SER E128:N,CA,OG,CB,C,O

[0855] SAVI8:PRO E129:N,CD,CA,CG,CB,C,O

[0856] SAVI8:SER E130:N,CA,OG,CB,C,O

[0857] SAVI8:PRO E131:N,CD,CA,CG,CB,C,O

[0858] SAVI8:SER E132:N,CA,OG,CB,C,O

[0859] SAVI8:ALA E133:N,CA,CB,C,O

[0860] SAVI8:THR E134:N,CA,CG2,OG1,CB,C,O

[0861] SAVI8:LEU E135:N,CA,CD2,CD1,CG,CB,C,O

[0862] SAVI8:GLU E136:N,CA,OE2,OE1,CD,CG,CB,C,O

[0863] SAVI8:GLN E137:N,CA,NE2,OE1,CD,CG,CB,C,O

[0864] SAVI8:ALA E138:N,CA,CB,C,O

[0865] SAVI8:VAL E139:N,CA,CG2,CG1,CB,C,O

[0866] SAVI8:ASN E140:N,CA,ND2,OD1,CG,CB,C,O

[0867] SAVI8:SER E141:N,CA,OG,CB,C,O

[0868] SAVI8:ALA E142:N,CA,CB,C,O

[0869] SAVI8:THR E143:N,CA,CG2,OG1,CB,C,O

[0870] SAVI8:SER E144:N,CA,OG,CB,C,O

[0871] SAVI8:VAL E149:N,CA,CG2,CG1,CB,C,O

[0872] SAVI8:VAL E150:N,CA,CG2,CG1,CB,C,O

[0873] SAVI8:SER E156:N,CA,OG,CB,C,O

[0874] SAVI8:GLY E157:N,CA,C,O

[0875] SAVI8:ALA E160:N,CA,CB,C,O

[0876] SAVI8:GLY E161:N,CA,C,O

[0877] SAVI8:SER E162:N,CA,OG,CB,C,O

[0878] SAVI8:ILE E165:N,CA,CD1,CG1,CB,CG2,C,O

[0879] SAVI8:SER E166:N,CA,OG,CB,C,O

[0880] SAVI8:TYR E167:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O

[0881] SAVI8:PRO E168:N,CD,CA,CG,CB,C,O

[0882] SAVI8:ARG E170:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0883] SAVI8:TYR E171:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O

[0884] SAVI8:ASN E173:N,CA,ND2,OD1,CG,CB,C,O

[0885] SAVI8:THR E180:N,CA,CG2,OG1,CB,C,O

[0886] SAVI8:ASP E181:N,CA,OD2,OD1,CG,CB,C,O

[0887] SAVI8:GLN E182:N,CA,NE2,OE1,CD,CG,CB,C,O

[0888] SAVI8:ASN E183:N,CA,ND2,OD1,CG,CB,C,O

[0889] SAVI8:ASN E184:N,CA,ND2,OD1,CG,CB,C,O

[0890] SAVI8:ASN E185:N,CA,ND2,OD1,CG,CB,C,O

[0891] SAVI8:ARG E186:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0892] SAVI8:ALA E187:N,CA,CB,C,O

[0893] SAVI8:SER E188:N,CA,OG,CB,C,O

[0894] SAVI8:SER E190:N,CA,OG,CB,C,O

[0895] SAVI8:GLN E191:N,CA,NE2,OE1,CD,CG,CB,C,O

[0896] SAVI8:TYR E192:N,CA,OH,CZ,CD2,CE2,CE1,CD1₁,CG,CB,C,O

[0897] SAVI8:ALA E200:N,CA,CB,C,O

[0898] SAVI8:VAL E203:N,CA,CG2,CG1,CB,C,O

[0899] SAVI8:ASN E204:N,CA,ND2,OD1,CG,CB,C,O

[0900] SAVI8:GLN E206:N,CA,NE2,OE1,CD,CG,CB,C,O

[0901] SAVI8:GLY E211:N,CA,C,O

[0902] SAVI8:SER E212:N,CA,OG,CB,C,O

[0903] SAVI8:THR E213:N,CA,CG2,OG1,CB,C,O

[0904] SAVI8:ALA E215:N,CA,CB,C,O

[0905] SAVI8:SER E216:N,CA,OG,CB,C,O

[0906] SAVI8:VAL E227:N,CA,CG2,CG1,CB,C,O

[0907] SAVI8:ALA E228:N,CA,CB,C,O

[0908] SAVI8:GLY E229:N,CA,C,O

[0909] SAVI8:ALA E230:N,CA,CB,C,O

[0910] SAVI8:THR E255:N,CA,CG2,OG1,CB,C,O

[0911] SAVI8:SER E256:N,CA,OG,CB,C,O

[0912] SAVI8:LEU E257:N,CA,CD2,CD1,CG,CB,C,O

[0913] SAVI8:GLY E258:N,CA,C,O

[0914] SAVI8:SER E259:N,CA,OG,CB,C,O

[0915] SAVI8:ASN E261:N,CA,ND2,OD1,CG,CB,C,O

[0916] SAVI8:LEU E262:N,CA,CD2,CD1,CG,CB,C,O

[0917] SAVI8:LEU E267:N,CA,CD2,CD1,CG,CB,C,O

[0918] SAVI8:VAL E268:N,CA,CG2,CG1,CB,C,O

[0919] SAVI8:ASN E269:N,CA,ND2,OD1,CG,CB,C,O

[0920] Subset SUB5B:

[0921] sub5bmole.list

[0922] Subset SUB5B:

[0923] SAVI8:E2-E4,E16,E19-E21,E23-E24,E28,E37,E41,E44-E45, E77-E81,E87-E88,

[0924] SAVI8:E90,E113-E114,E117-E118,E120-E121,E145-E148,E169,E172,E174-E176,

[0925] SAVI8:E193-E196,E198-E199,E214,E231-E234,E236,E243,E247,E250,E253-E254,

[0926] SAVI8:E260,E263-E266,E270-E273,M276H-M277H

[0927] sub5batom.list

[0928] Subset SUB5B:

[0929] SAVI8:GLN E2:N,CA,NE2,OE1,CD,CG,CB,C,O

[0930] SAVI8:SER E3:N,CA,OG,CB,C,O

[0931] SAVI8:VAL E4:N,CA,CG2,CG1,CB,C,O

[0932] SAVI8:ALA E16:N,CA,CB,C,O

[0933] SAVI8:ARG E19:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0934] SAVI8:GLY E20:N,CA,C,O

[0935] SAVI8:LEU E21:N,CA,CD2,CD1,CG,CB,C,O

[0936] SAVI8:GLY.E23:N,CA,C,O

[0937] SAVI8:SER E24:N,CA,OG,CB,C,O

[0938] SAVI8:VAL E28:N,CA,CG2,CG1,CB,C,O

[0939] SAVI8:SER E37:N,CA,OG,CB,C,O

[0940] SAVI8:ASP E41:N,CA,OD2,OD1,CG,CB,C,O

[0941] SAVI8:ILE E44:N,CA,CD1,CG1,CB,CG2,C,O

[0942] SAVI8:ARG E45:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0943] SAVI8:ASN E77:N,CA,ND2,OD1,CG,CB,C,O

[0944] SAVI8:SER E78:N,CA,OG,CB,C,O

[0945] SAVI8:ILE E79:N,CA,CD1,CG1,CB,CG2,C,O

[0946] SAVI8:GLY E80:N,CA,C,O

[0947] SAVI8:VAL E81:N,CA,CG2,CG1,CB,C,O

[0948] SAVI8:SER E87:N,CA,OG,CB,C,O

[0949] SAVI8:ALA E88:N,CA,CB,C,O

[0950] SAVI8:LEU E90:N,CA,CD2,CD1,CG,CB,C,O

[0951] SAVI8:TRP E113:N,CA,CD2,CE2,NE1,CD1,CG,CE3,CZ3,CH2,CZ2,CB,C,O

[0952] SAVI8:ALA E114:N,CA,CB,C,O

[0953] SAVI8:ASN E117:N,CA,ND2,OD1,CG,CB,C,O

[0954] SAVI8:GLY E118:N,CA,C,O

[0955] SAVI8:HIS E120:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[0956] SAVI8:VAL E121:N,CA,CG2,CG1,CB,C,O

[0957] SAVI8:ARG E145:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0958] SAVI8:GLY E146:N,CA,C,O

[0959] SAVI8:VAL E147:N,CA,CG2,CG1,CB,C,O

[0960] SAVI8:LEU E148:N,CA,CD2,CD1,CG,CB,C,O

[0961] SAVI8:ALA E169:N,CA,CB,C,O

[0962] SAVI8:ALA E172:N,CA,CB,C,O

[0963] SAVI8:ALA E174:N,CA,CB,C,O

[0964] SAVI8:MET E175:N,CA,CE,SD,CG,CB,C,O

[0965] SAVI8:ALA E176:N,CA,CB,C,O

[0966] SAVI8:GLY E193:N,CA,C,O

[0967] SAVI8:ALA E194:N,CA,CB,C,O

[0968] SAVI8:GLY E195:N,CA,C,O

[0969] SAVI8:LEU E196:N,CA,CD2,CD1,CG,CB,C,O

[0970] SAVI8:ILE E198:N,CA,CD1,CG1,CB,CG2,C,O

[0971] SAVI8:VAL E199:N,CA,CG2,CG1,CB,C,O

[0972] SAVI8:TYR E214:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O

[0973] SAVI8:ALA E231:N,CA,CB,C,O

[0974] SAVI8:ALA E232:N,CA,CB,C,O

[0975] SAVI8:LEU E233:N,CA,CD2,CD1,CG,CB,C,O

[0976] SAVI8:VAL E234:N,CA,CG2,CG1,CB,C,O

[0977] SAVI8:GLN E236:N,CA,NE2,OE1,CD,CG,CB,C,O

[0978] SAVI8:ASN E243:N,CA,ND2,OD1,CG,CB,C,O

[0979] SAVI8:ARG E247:N,CA,NH2,NH1,CZ,NE,CD,CG,CB,C,O

[0980] SAVI8:LEU E250:N,CA,CD2,CD1,CG,CB,C,O

[0981] SAVI8:THR E253:N,CA,CG2,OG1,CB,C,O

[0982] SAVI8:ALA E254:N,CA,CB,C,O

[0983] SAVI8:THR E260:N,CA,CG2,OG1,CB,C,O

[0984] SAVI8:TYR E263:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O

[0985] SAVI8:GLY E264:N,CA,C,O

[0986] SAVI8:SER E265:N,CA,OG,CB,C,O

[0987] SAVI8:GLY E266:N,CA,C,O

[0988] SAVI8:ALA E270:N,CA,CB,C,O

[0989] SAVI8:GLU E271:N,CA,OE2,OE1,CD,CG,CB,C,O

[0990] SAVI8:ALA E272:N,CA,CB,C,O

[0991] SAVI8:ALA E273:N,CA,CB,C,O

[0992] SAVI8:ION M276H:CA

[0993] SAVI8:ION M277H:CA

[0994] Subset ACTSITE:

[0995] actsitemole.list

[0996] Subset ACTSITE:

[0997] SAVI8:E29-E35,E48-E51,E54,E58-E72,E91-E102,E106-E107,E110,E123-E127,

[0998] SAVI8: E151-E155,E177-E179,E189,E201-E202,E205,E207-E210,E217-E226

[0999] actsiteatom.list

[1000] Subset ACTSITE:

[1001] SAVI8:ALA E29:N,CA,CB,C,O

[1002] SAVI8:VAL E30:N,CA,CG2,CG1,CB,C,O

[1003] SAVI8:LEU E31:N,CA,CD2,CD1,CG,CB,C,O

[1004] SAVI8:ASP E32:N,CA,OD2,OD1,CG,CB,C,O

[1005] SAVI8:THR E33:N,CA,CG2,OG1,CB,C,O

[1006] SAVI8:GLY E34:N,CA,C,O

[1007] SAVI8:ILE E35:N,CA,CD1,CG1,CB,CG2,C,O

[1008] SAVI8:ALA E48:N,CA,CB,C,O

[1009] SAVI8:SER E49:N,CA,OG,CB,C,O

[1010] SAVI8:PHE E50:N,CA,CD2,CE2,CZ,CE1,CD1,CG,CB,C,O

[1011] SAVI8:VAL E51:N,CA,CG2,CG1,CB,C,O

[1012] SAVI8:GLU E54:N,CA,OE2,OE1,CD,CG,CB,C,O

[1013] SAVI8:THR E58:N,CA,CG2,OG1,CB,C,O

[1014] SAVI8:GLN E59:N,CA,NE2,OE1,CD,CG,CB,C,O

[1015] SAVI8:ASP E60:N,CA,OD2,OD1,CG,CB,C,O

[1016] SAVI8:GLY E61:N,CA,C,O

[1017] SAVI8:ASN E62:N,CA,ND2,OD1,CG,CB,C,O

[1018] SAVI8:GLY E63:N,CA,C,O

[1019] SAVI8:HIS E64:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[1020] SAVI8:GLY E65:N,CA,C,O

[1021] SAVI8:THR E66:N,CA,CG2,OG1,CB,C,O

[1022] SAVI8:HIS E67:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[1023] SAVI8:VAL E68:N,CA,CG2,CG1,CB,C,O

[1024] SAVI8:ALA E69:N,CA,CB,C,O

[1025] SAVI8:GLY E70:N,CA,C,O

[1026] SAVI8:THR E71:N,CA,CG2,OG1,CB,C,O

[1027] SAVI8:ILE E72:N,CA,CD1,CG1,CB,CG2,C,O

[1028] SAVI8:TYR E91:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O

[1029] SAVI8:ALA E92:N,CA,CB,C,O

[1030] SAVI8:VAL E93:N,CA,CG2,CG1,CB,C,O

[1031] SAVI8:LYS E94:N,CA,NZ,CE,CD,CG,CB,C,O

[1032] SAVI8:VAL E95:N,CA,CG2,CG1,CB,C,O

[1033] SAVI8:LEU E96:N,CA,CD2,CD1,CG,CB,C,O

[1034] SAVI8:GLY E97:N,CA,C,O

[1035] SAVI8:ALA E98:N,CA,CB,C,O

[1036] SAVI8:SER E99:N,CA,OG,CB,C,O

[1037] SAVI8:GLY E100:N,CA,C,O

[1038] SAVI8:SER E101:N,CA,OG,CB,C,O

[1039] SAVI8:GLY E102:N,CA,C,O

[1040] SAVI8:SER E106:N,CA,OG,CB,C,O

[1041] SAVI8:ILE E107:N,CA,CD1,CG1,CB,CG2,C,O

[1042] SAVI8:GLY E110:N,CA,C,O

[1043] SAVI8:ASN E123:N,CA,ND2,OD1,CG,CB,C,O

[1044] SAVI8:LEU E124:N,CA,CD2,CD1,CG,CB,C,O

[1045] SAVI8:SER E125:N,CA,OG,CB,C,O

[1046] SAVI8:LEU E126:N,CA,CD2,CD1,CG,CB,C,O

[1047] SAVI8:GLY E127:N,CA,C,O

[1048] SAVI8:ALA E151:N,CA,CB,C,O

[1049] SAVI8:ALA E152:N,CA,CB,C,O

[1050] SAVI8:SER E153:N,CA,OG,CB,C,O

[1051] SAVI8:GLY E154:N,CA,C,O

[1052] SAVI8:ASN E155:N,CA,ND2,OD1,CG,CB,C,O

[1053] SAVI8:VAL E177:N,CA,CG2,CG1,CB,C,O

[1054] SAVI8:GLY E178:N,CA,C,O

[1055] SAVI8:ALA E179:N,CA,CB,C,O

[1056] SAVI8:PHE E189:N,CA,CD2,CE2,CZ, CE1,CD1,CG,CB,C,O

[1057] SAVI8:PRO E201:N,CD,CA,CG,CB,C,O

[1058] SAVI8:GLY E202:N,CA,C,O

[1059] SAVI8:VAL E205:N,CA,CG2,CG1,CB,C,O

[1060] SAVI8:SER E207:N,CA,OG,CB,C,O

[1061] SAVI8:THR E208:N,CA,CG2,OG1,CB,C,O

[1062] SAVI8:TYR E209:N,CA,OH,CZ,CD2,CE2,CE1,CD1,CG,CB,C,O

[1063] SAVI8:PRO E210:N,CD,CA,CG,CB,C,O

[1064] SAVI8:LEU E217:N,CA,CD2,CD1,CG,CB,C,O

[1065] SAVI8:ASN E218:N,CA,ND2,OD1,CG,CB,C,O

[1066] SAVI8:GLY E219:N,CA,C,O

[1067] SAVI8:THR E220:N,CA,CG2,OG1,CB,C,O

[1068] SAVI8:SER E221:N,CA,OG,CB,C,O

[1069] SAVI8:MET E222:N,CA,CE,SD,CG,CB,C,O

[1070] SAVI8:ALA E223:N,CA,CB,C,O

[1071] SAVI8:THR E224:N,CA,CG2,OG1,CB,C,O

[1072] SAVI8:PRO E225:N,CD,CA,CG,CB,C,O

[1073] SAVI8:HIS E226:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[1074] Subset RESTx:

[1075] restxmole.list

[1076] Subset RESTx:

[1077] NEWMODEL:E5,E13-E14,E22,E38-E40,E42,E73-E76,E82-E86,E103-E105,

[1078] NEWMODEL:E108,E122,E133-E135,E137-E140,E149-E150,E173,E204,E206,

[1079] NEWMODEL:E211-E213,E215-E216,E227-E229,E258,E269

[1080] restxatom.list

[1081] Subset RESTx:

[1082] NEWMODEL:PRO E5:N,CD,CA,CG,CB,C,O

[1083] NEWMODEL:ALA E13:N,CA,CB,C,O

[1084] NEWMODEL:PRO E14:N,CD,CA,CG,CB,C,O

[1085] NEWMODEL:THR E22:N,CA,CG2,OG1,CB,C,O

[1086] NEWMODEL:THR E38:N,CA,CG2,OG1,CB,C,O

[1087] NEWMODEL:HIS E39:N,CA,CD2,NE2,CE1,ND1,CG,CB,C,O

[1088] NEWMODEL:PRO E40:N,CD,CA,CG,CB,C,O

[1089] NEWMODEL:LEU E42:N,CA,CD2,CD1,CG,CB,C,O

[1090] NEWMODEL:ALA E73:N,CA,CB,C,O

[1091] NEWMODEL:ALA E74:N,CA,CB,C,O

[1092] NEWMODEL:LEU E75:N,CA,CD2,CD1,CG,CB,C,O

[1093] NEWMODEL:ASN E76:N,CA,ND2,OD1,CG,CB,C,O

[1094] NEWMODEL:LEU E82:N,CA,CD2,CD1,CG,CB,C,O

[1095] NEWMODEL:GLY E83:N,CA,C,O

[1096] NEWMODEL:VAL E84:N,CA,CG2,CG1,CB,C,O

[1097] NEWMODEL:ALA E85:N,CA,CB,C,O

[1098] NEWMODEL:PRO E86:N,CD,CA,CG,CB,C,O

[1099] NEWMODEL:SER E103:N,CA,OG,CB,C,O

[1100] NEWMODEL:VAL E104:N,CA,CG2,CG1,CB,C,O

[1101] NEWMODEL:SER E105:N,CA,OG,CB,C,O

[1102] NEWMODEL:ALA E108:N,CA,CB,C,O

[1103] NEWMODEL:ALA E122:N,CA,CB,C,O

[1104] NEWMODEL:ALA E133:N,CA,CB,C,O

[1105] NEWMODEL:THR E134:N,CA,CG2,OG1,CB,C,O

[1106] NEWMODEL:LEU E135:N,CA,CD2,CD1,CG,CB,C,O

[1107] NEWMODEL:GLN E137:N,CA,NE2,OE1,CD,CG,CB,C,O

[1108] NEWMODEL:ALA E138:N,CA,CB,C,O

[1109] NEWMODEL:VAL E139:N,CA,CG2,CG1,CB,C,O

[1110] NEWMODEL:ASN E140:N,CA,ND2,CD1,CG,CB,C,O

[1111] NEWMODEL:VAL E149:N,CA,CG2,CG1,CB,C,O

[1112] NEWMODEL:VAL E150:N,CA,CG2,CG1,CB,C,O

[1113] NEWMODEL:ASN E173:N,CA,ND2,OD1,CG,CB,C,O

[1114] NEWMODEL:ASN E204:N,CA,ND2,OD1,CG,CB,C,O

[1115] NEWMODEL:GLN E206:N,CA,NE2,OE1,CD,CG,CB,C,O

[1116] NEWMODEL:GLY E211:N,CA,C,O

[1117] NEWMODEL:SER E212:N,CA,OG,CB,C,O

[1118] NEWMODEL:THR E213:N,CA,CG2,OG1,CB,C,O

[1119] NEWMODEL:ALA E215:N,CA,CB,C,O

[1120] NEWMODEL:SER E216:N,CA,OG,CB,C,O

[1121] NEWMODEL:VAL E227:N,CA,CG2,CG1,CB,C,O

[1122] NEWMODEL:ALA E228:N,CA,CB,C,O

[1123] NEWMODEL:GLY E229:N,CA,C,O

[1124] NEWMODEL:GLY E258:N,CA,CO

[1125] NEWMODEL:ASN E269:N,CA,ND2,OD1,CG,CB,C,O

[1126] Suitable Substitutions in PD498 for Addition of Carboxylic Acid Attachment Groups (—COOH)

[1127] The 3D structure of PD498 was modeled as described in Example 1. Suitable locations for addition of carboxylic attachment groups (Aspartatic acids and Glutamic acids) were found as follows. The procedure described in Example 1 was followed. The commands performed in Insight (BIOSYM) are shown in the command files makeDEzone.bcl and makeDEzone2.bcl below:

[1128] Conservative Substutitions:

[1129] makeDEzone.bcl

[1130] Delete Subset *

[1131] Color Molecule Atoms * Specified Specification255,0,255

[1132] Zone Subset ASP :asp:od* Static monomer/residue 10 Color_Subset 255,255,0

[1133] Zone Subset GLU :glu:oe* Static monomer/residue 10 Color_Subset 255,255,0

[1134] #NOTE: editnextline C-terminal residue number according to the protein

[1135] Zone Subset CTERM :280:0 Static monomer/residue 10 Color_Subset 255,255,0

[1136] #NOTE: editnextline ACTSITE residues according to the protein

[1137] Zone Subset ACTSITE :39,72,226 Static monomer/residue 8

[1138] Color_Subset 255,255,0

[1139] Combine Subset ALLZONE Union ASP GLU

[1140] Combine Subset ALLZONE Union ALLZONE CTERM

[1141] Combine Subset ALLZONE Union ALLZONE ACTSITE

[1142] #NOTE: editnextline object name according to the protein

[1143] Combine Subset REST Difference PD498FINALMODEL ALLZONE

[1144] List Subset REST Atom output_File restatom.list

[1145] List Subset REST monomer/residue Output_File restmole.list

[1146] Color Molecule Atoms ACTSITE Specified Specification 255,0,0

[1147] List Subset ACTSITE Atom Output_File actsiteatom.list

[1148] List Subset ACTSITE monomer/residue Output_File actsitemole.list

[1149] #

[1150] Zone Subset REST5A REST Static Monomer/Residue 5- Color_Subset

[1151] Combine Subset SUB5A Difference REST5A ACTSITE

[1152] Combine Subset SUB5B Difference SUB5A REST

[1153] Color Molecule Atoms SUB5B Specified Specification 255,255,255

[1154] List Subset SUB5B Atom Output_File sub5batom.list

[1155] List Subset SUB5B monomer/residue Output_File sub5bmole.list

[1156] #Now identify sites for asn→asp & gln→glu substitutions and . . .

[1157] #continue with makezone2.bcl.

[1158] #Use grep command to identify asn/gln in restatom.list . . .

[1159] #sub5batom.list & accsiteatom.list

[1160] Comments:

[1161] The subset REST contains Gln33 and Asn245, SUB5B contains Gln12, Gln126, Asn209, Gln242, Asn246, Gln248 and Asn266, all of which are solvent exposed.

[1162] The substitutions Q12E or Q12D, Q33E or Q33D, Q126E or Q126D, N209D or N209E, Q242E or Q242D, N245D or N245E, N246D or N246E, Q248E or Q248D and N266D or N266E are identified in PD498 as sites for mutagenesis within the scope of this invention. Residues are substituted below in section 2, and further analysis done:

[1163] Non-conservative Substitutions:

[1164] makeDEzone2.bcl

[1165] #sourcefile makezone2.bcl Claus von der Osten 961128

[1166] #

[1167] #having scanned lists (grep gln/asn command) and identified

[1168] sites for . . .

[1169] #asn→asp & gln→glu substitutions

[1170] #NOTE: editnextline object name according to protein

[1171] Copy Object -To_Clipboard -Displace PD498FINALMODEL newmodel Biopolymer

[1172] #NOTE: editnextline object name according to protein

[1173] Blank Object On PD498FINALMODEL

[1174] #NOTE: editnextlines with asn→asp & gln→glu positions

[1175] Replace Residue newmodel:33 glu L

[1176] Replace Residue newmodel:245 asp L

[1177] Replace Residue newmodel:12 glu L

[1178] Replace Residue newmodel:126 glu L

[1179] Replace Residue newmodel:209 asp L

[1180] Replace Residue newmodel:242 glu L

[1181] Replace Residue newmodel:246 asp L

[1182] Replace Residue newmodel:248 glu L

[1183] Replace Residue newmodel:266 asp L

[1184] #

[1185] #Now repeat analysis done prior to asn→asp & gln→glu, . . .

[1186] #now including introduced asp & glu

[1187] Color Molecule Atoms newmodel Specified Specification 255,0,255

[1188] Zone Subset ASPx newmodel:Asp:od* Static monomer/residue 10

[1189] Color_Subset 255,255,0

[1190] Zone Subset GLUx newmodel:glu:oe* Static monomer/residue 10

[1191] Color_Subset 255,255,0

[1192] #NOTE: editnextline C-terminal residue number according to the protein

[1193] Zone Subset CTERMx newmodel:280: O Static monomer/residue 10

[1194] Color_Subset 255,255,0

[1195] #NOTE. editnextline ACTSITEx residues according to the protein

[1196] Zone Subset ACTSITEx newmodel:39,72,226 Static monomer/residue

[1197] 8 Color_Subset 255,255,0

[1198] Combine Subset ALLZONEx Union ASPx GLUx

[1199] Combine Subset ALLZONEx Union ALLZONEx CTERMX

[1200] Combine Subset ALLZONEx Union ALLZONEx ACTSITEx

[1201] Combine Subset RESTx Difference newmodel ALLZONEx

[1202] List Subset RESTx Atom output_File restxatom.list

[1203] List Subset RESTx monomer/residue Output_File restxmole.list

[1204] #

[1205] Color Molecule Atoms ACTSITEx Specified Specification 255,0,0

[1206] List Subset ACTSITEx Atom Output_File actsitexatom.list

[1207] List Subset ACTSITEx monomer/residue Output_File actsitexmole.list

[1208] #

[1209] #read restxatom.list or restxmole.list to identify sites for

[1210] (not_gluasp)→gluasp . . .

[1211] #subst. if needed

[1212] Comments:

[1213] The subset RESTx contains only two residues: A233 and G234, none of which are solvent exposed. No further mutagenesis is required to obtain complete protection of the surface. However, it may be necessary to remove some of the reactive carboxylic groups in the active site region to ensure access to the active site of PD498. Acidic residues within the subset ACTSITE are: D39, D58, D68 and D106. Of these only the two latter are solvent exposed and D39 is a functional residue. The mutations D68N, D68Q, D106N and D106Q were found suitable according to the present invention.

[1214] Relevant data for Example 3: Solvent accessibility data for PD498MODEL: see Example 1 above.

[1215] Subset REST:

[1216] restmole.list

[1217] Subset REST:

[1218] PD498FINALMODEL:10-11,33-35,54-55,129-130,221,233-234,236,240,243,

[1219] PD498FINALMODEL:245,262,264-265

[1220] restatom.list

[1221] Subset REST:

[1222] PD498FINALMODEL:ALA 10:N,CA,C,O,CB

[1223] PD498FINALMODEL:TYR 11:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1224] PD498FINALMODEL:GLN 33:N,CA,C,O,CB,CG,CD,OE1,NE2

[1225] PD498FINALMODEL:THR 34:N,CA,C,O,CB,OG1,CG2

[1226] PD498FINALMODEL:VAL 35:N,CA,C,O,CB,CG1,CG2

[1227] PD498FINALMODEL:ILE 54:N,CA,C,O,CB,CG1,CG2,CD1

[1228] PD498FINALMODEL:LYS 55:N,CA,C,O,CB,CG,CD,CE,NZ

[1229] PD498FINALMODEL:LYS 129:N,CA,C,O,CB,CG,CD,CE,NZ

[1230] PD498FINALMODEL:VAL 130:N,CA,C,O,CB,CG1,CG2

[1231] PD498FINALMODEL:TYR 221:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1232] PD498FINALMODEL:ALA 233:N,CA,C,O,CB

[1233] PD498FINALMODEL:GLY 234:N,CA,C,O

[1234] PD498FINALMODEL:ALA 236:N,CA,C,O,CB

[1235] PD498FINALMODEL:ALA 240:N,CA,C,O,CB

[1236] PD498FINALMODEL:GLY 243:N,CA,C,O

[1237] PD498FINALMODEL:ASN 245:N,CA,C,O,CB,CG,OD1,ND2

[1238] PD498FINALMODEL:GLY 262:N,CA,C,O

[1239] PD498FINALMODEL:GLY 264:N,CA,C,O

[1240] PD498FINALMODEL:THR 265:N,CA,C,O,CB,OG1,CG2

[1241] Subset SUB5B:

[1242] sub5bmole.list

[1243] Subset SUB5B:

[1244] PD498FINALMODEL:6-9,12-13,3-1-32,51-53, 56,81,93-94,97-99,122,126-128,

[1245] PD498FINALMODEL:131,155-157,159,197-199,209,211,219-220,232,235,

[1246] PD498FINALMODEL:237-239,241-242,244,246-249, 253,260-261,263,266-268

[1247] sub5batom.list

[1248] Subset SUB5B:

[1249] PD498FINALMODEL:PRO 6:N,CA,CD,C,O,CB,CG

[1250] PD498FINALMODEL:TYR 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1251] PD498FINALMODEL:TYR 8:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1252] PD498FINALMODEL:SER 9:N,CA,C,O,CB,OG

[1253] PD498FINALMODEL:GLN 12:N,CA,C,O,CB,CG,CD,OE1,NE2

[1254] PD498FINALMODEL:TYR 13:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1255] PD498FINALMODEL:SER 31:N,CA,C,O,CB,OG

[1256] PD498FINALMODEL:THR 32:N,CA,C,O,CB,OG1,CG2

[1257] PD498FINALMODEL:ARG 51:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1258] PD498FINALMODEL:LYS 52:N,CA,C,O,CB,CG,CD,CE,NZ

[1259] PD498FINALMODEL:VAL 53:N,CA,C,O,CB,CG1,CG2

[1260] PD498FINALMODEL:GLY 56:N,CA,C,O

[1261] PD498FINALMODEL:ALA 81:N,CA,C,O,CB

[1262] PD498FINALMODEL:MET 93:N,CA,C,O,CB,CG,SD,CE

[1263] PD498FINALMODEL:ALA 94:N,CA,C,O,CB

[1264] PD498FINALMODEL:THR 97:N,CA,C,O,CB,OG1,CG2

[1265] PD498FINALMODEL:LYS 98:N,CA,C,O,CB,CG,CD,CE,NZ

[1266] PD498FINALMODEL:ILE 99:N,CA,C,O,CB,CG1,CG2,CD1

[1267] PD498FINALMODEL:TYR 122:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1268] PD498FINALMODEL:GLN 126:N,CA,C,O,CB,CG,CD,OE1,NE2

[1269] PD498FINALMODEL:GLY 127:N,CA,C,O

[1270] PD498FINALMODEL:ALA 128:N,CA,C,O,CB

[1271] PD498FINALMODEL:LEU 131:N,CA,C,O,CB,CG,CD1,CD2

[1272] PD498FINALMODEL:GLY 155:N,CA,C,O

[1273] PD498FINALMODEL:ALA 156:N,CA,C,O,CB

[1274] PD498FINALMODEL:VAL 157:N,CA,C,O,CB,CG1,CG2

[1275] PD498FINALMODEL:VAL 159:N,CA,C,O,CB,CG1,CG2

[1276] PD498FINALMODEL:TYR 197:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1277] PD498FINALMODEL:GLY 198:N,CA,C,O

[1278] PD498FINALMODEL:THR 199:N,CA,C,O,CB,OG1,CG2

[1279] PD498FINALMODEL:ASN 209:N,CA,C,O,CB,CG,OD1,ND2

[1280] PD498FINALMODEL:ALA 211:N,CA,C,O,CB

[1281] PD498FINALMODEL:TYR 219:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1282] PD498FINALMODEL:SER 220:N,CA,C,O,CB,OG

[1283] PD498FINALMODEL:VAL 232:N,CA,C,O,CB,CG1,CG2

[1284] PD498FINALMODEL:LEU 235:N,CA,C,O,CB,CG,CD1,CD2

[1285] PD498FINALMODEL:ALA 237:N,CA,C,O,CB

[1286] PD498FINALMODEL:LEU 238:N,CA,C,O,CB,CG,CD1,CD2

[1287] PD498FINALMODEL:LEU 239:N,CA,C,O,CB,CG,CD1,CD2

[1288] PD498FINALMODEL:SER 241:N,CA,C,O,CB,OG

[1289] PD498FINALMODEL:GLN 242:N,CA,C,O,CB,CG,CD,OE1,NE2

[1290] PD498FINALMODEL:LYS 244:N,CA,C,O,CB,CG,CD,CE,NZ

[1291] PD498FINALMODEL:ASN 246:N,CA,C,O,CB,CG,OD1,ND2

[1292] PD498FINALMODEL:VAL 247:N,CA,C,O,CB,CG1,CG2

[1293] PD498FINALMODEL:GLN 248:N,CA,C,O,CB,CG,CD,OE1,NE2

[1294] PD498FINALMODEL:ILE 249:N,CA,C,O,CB,CG1,CG2,CD1

[1295] PD498FINALMODEL:ILE 253:N,CA,C,O,CB,CG1,CG2,CD1

[1296] PD498FINALMODEL:ILE 260:N,CA,C,O,CB,CG1,CG2,CD1

[1297] PD498FINALMODEL:SER 261:N,CA,C,O,CB,OG

[1298] PD498FINALMODEL:THR 263:N,CA,C,O,CB,OG1,CG2

[1299] PD498FINALMODEL:ASN 266:N,CA,C,O,CB,CG,OD1,ND2

[1300] PD498FINALMODEL:PHE 267:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1301] PD498FINALMODEL:LYS 268:N,CA,C,O,CB,CG,CD,CE,NZ

[1302] Subset ACTSITE:

[1303] actsitemole.list

[1304] Subset ACTSITE:

[1305] PD498FINALMODEL:36-42,57-60,66-80,100-110,115-116,119,132-136,160-164,

[1306] PD498FINALMODEL:182-184,194,206-207,210,212-215,222-231

[1307] actsiteatom.list

[1308] Subset ACTSITE:

[1309] PD498FINALMODEL:ALA 36:N,CA,C,O,CB

[1310] PD498FINALMODEL:VAL 37:N,CA,C,O,CB,CG1,CG2

[1311] PD498FINALMODEL:LEU 38:N,CA,C,O,CB,CG,CD1,CD2

[1312] PD498FINALMODEL:ASP 39:N,CA,C,O,CB,CG,OD1,OD2

[1313] PD498FINALMODEL:SER 40:N,CA,C,O,CB,OG

[1314] PD498FINALMODEL:GLY 41:N,CA,C,O

[1315] PD498FINALMODEL:VAL 42:N,CA,C,O,CB,CG1,CG2

[1316] PD498FINALMODEL:TYR

[1317] 57:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1318] PD498FINALMODEL:ASP 58:N,CA,C,O,CB,CG,OD1,OD2

[1319] PD498FINALMODEL:PHE

[1320] 59:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1321] PD498FINALMODEL:ILE 60:N,CA,C,O,CB,CG1,CG2,CD1

[1322] PD498FINALMODEL:PRO 66:N,CA,CD,C,O,CB,CG

[1323] PD498FINALMODEL:MET 67:N,CA,C,O,CB,CG,SD,CE

[1324] PD498FINALMODEL:ASP 68:N,CA,C,O,CB,CG,OD1,OD2

[1325] PD498FINALMODEL:LEU 69:N,CA,C,O,CB,CG,CD1,CD2

[1326] PD498FINALMODEL:ASN 70:N,CA,C,O,CB,CG,OD1,ND2

[1327] PD498FINALMODEL:GLY 71:N,CA,C,O

[1328] PD498FINALMODEL:HIS 72:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1329] PD498FINALMODEL:GLY 73:N,CA,C,O

[1330] PD498FINALMODEL:THR 74:N,CA,C,O,CB,OG1,CG2

[1331] PD498FINALMODEL:HIS 75:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1332] PD498FINALMODEL:VAL 76:N,CA,C,O,CB,CG1,CG2

[1333] PD498FINALMODEL:ALA 77:N,CA,C,O,CB

[1334] PD498FINALMODEL:GLY 78:N,CA,C,O

[1335] PD498FINALMODEL:THR 79:N,CA,C,O,CB,OG1,CG2

[1336] PD498FINALMODEL:VAL 80:N,CA,C,O,CB,CG1,CG2

[1337] PD498FINALMODEL:LEU 100:N,CA,C,O,CB,CG,CD1,CD2

[1338] PD498FINALMODEL:ALA 101:N,CA,C,O,CB

[1339] PD498FINALMODEL:VAL 102:N,CA,C,O,CB,CG1,CG2

[1340] PD498FINALMODEL:ARG 103:N,CA,C,O,CB,

[1341] CG,CD,NE,CZ,NH1,NH2

[1342] PD498FINALMODEL:VAL 104:N,CA,C,O,CB,CG1,CG2

[1343] PD498FINALMODEL:LEU 105:N,CA,C,O,CB,CG,CD1,CD2

[1344] PD498FINALMODEL:ASP 106:N,CA,C,O,CB,CG,OD1,OD2

[1345] PD498FINALMODEL:ALA 107:N,CA,C,O,CB

[1346] PD498FINALMODEL:ASN 108:N,CA,C,O,CB,CG,OD1,ND2

[1347] PD498FINALMODEL:GLY 109:N,CA,C,O

[1348] PD498FINALMODEL:SER 110:N,CA,C,O,CB,OG

[1349] PD498FINALMODEL:SER 115:N,CA,C,O,CB,OG

[1350] PD498FINALMODEL:ILE 116:N,CA,C,O,CB,

[1351] CG1,CG2,CD1

[1352] PD498FINALMODEL:GLY 119:N,CA,C,O

[1353] PD498FINALMODEL:ASN 132:N,CA,C,O,CB,CG,OD1,ND2

[1354] PD498FINALMODEL:LEU 133:N,CA,C,O,CB,CG,CD1,CD2

[1355] PD498FINALMODEL:SER 134:N,CA,C,O,CB,OG

[1356] PD498FINALMODEL:LEU 135:N,CA,C,O,CB,CG,CD1,CD2

[1357] PD498FINALMODEL:GLY 136:N,CA,C,O

[1358] PD498FINALMODEL:ALA 160:N,CA,C,O,CB

[1359] PD498FINALMODEL:ALA 161:N,CA,C,O,CB

[1360] PD498FINALMODEL:ALA 162:N,CA,C,O,CB

[1361] PD498FINALMODEL:GLY 163:N,CA,C,O

[1362] PD498FINALMODEL:ASN 164:N,CA,C,O,CB,CG,OD1,ND2

[1363] PD498FINALMODEL:VAL 182:N,CA,C,O,CB,CG1,CG2

[1364] PD498FINALMODEL:GLY 183:N,CA,C,O

[1365] PD498FINALMODEL:ALA 184:N,CA,C,O,CB

[1366] PD498FINALMODEL:PHE 194:N,CA,C,O,CB,

[1367] CG,CD1,CD2,CE1,CE2,CZ

[1368] PD498FINALMODEL:PRO 206:N,CA,CD,C,O,CB,CG

[1369] PD498FINALMODEL:GLY 207:N,CA,C,O

[1370] PD498FINALMODEL:ILE 210:N,CA,C,O,CB,

[1371] CG1,CG2,CD1

[1372] PD498FINALMODEL:SER 212:N,CA,C,O,CB,OG

[1373] PD498FINALMODEL:THR 213:N,CA,C,O,CB,OG1,CG2

[1374] PD498FINALMODEL:VAL 214:N,CA,C,O,CB,CG1,CG2

[1375] PD498FINALMODEL:PRO 215:N,CA,CD,C,O,CB,CG

[1376] PD498FINALMODEL:MET 222:N,CA,C,O,CB,CG,SD,CE

[1377] PD498FINALMODEL:SER 223:N,CA,C,O,CB,OG

[1378] PD498FINALMODEL:GLY 224:N,CA,C,O

[1379] PD498FINALMODEL:THR 225:N,CA,C,O,CB,OG1,CG2

[1380] PD498FINALMODEL:SER 226:N,CA,C,O,CB,OG

[1381] PD498FINALMODEL:MET 227:N,CA,C,O,CB,CG,SD,CE

[1382] PD498FINALMODEL:ALA 228:N,CA,C,O,CB

[1383] PD498FINALMODEL:SER 229:N,CA,C,O,CB,OG

[1384] PD498FINALMODEL:PRO 230:N,CA,CD,C,O,CB,CG

[1385] PD498FINALMODEL:HIS 231:N,CA,C,O,CB,

[1386] CG,ND1,CD2,CE1,NE2

[1387] Subset RESTx:

[1388] restxmole.list

[1389] Subset RESTX:

[1390] NEWMODEL:233-234

[1391] restxatom.list

[1392] Subset RESTX:

[1393] NEWMODEL:ALA 233:N,CA,C,O,CB

[1394] NEWMODEL:GLY 234:N,CA,C,O

[1395] Suitable Substitutions in the Arthromyces RamosusPeroxidase for Addition of Carboxylic Acid Attachment Groups (—COOH)

[1396] Suitable locations for addition of carboxylic attachment groups (Aspartatic acids and Glutamic acids) in a non-hydrolytic enzyme, Arthromyces ramosusperoxidase were found as follows.

[1397] The 3D structure of this oxido-reductase is available in the Brookhaven Databank as 1arp.pdb. This A. ramosusperoxidase contains 344 amino acid residues. The first eight residues are not visible in the X-ray structure: QGPGGGGG, and N143 is glycosylated. The procedure described in Example 1 was followed.

[1398] The amino acid sequence of Arthromyces ramosusPeroxidase (E.C.1.11.1.7) is shown in SEQ ID NO 4.

[1399] The commands performed in Insight (BIOSYM) are shown in the command files makeDEzone.bcl and makeDEzone2.bcl below. The C-terminal residue is P344, the ACTSITE is defined as the heme group and the two histidines coordinating it (H56 & H184).

[1400] Conservative Substitutions:

[1401] makeDEzone.bcl

[1402] Delete Subset *

[1403] Color Molecule Atoms * Specified Specification 255,0,255

[1404] Zone Subset ASP :asp:od* Static monomer/residue 10 Color_Subset 255,255,0

[1405] Zone Subset GLU :glu:oe* Static monomer/residue 10 Color_Subset 255,255,0

[1406] #NOTE: editnextline C-terminal residue number according to the

[1407] 40 protein

[1408] Zone Subset CTERM :344:0 Static monomer/residue 10 Color_Subset 255,255,0

[1409] #NOTE: editnextline ACTSITE residues according to the protein

[1410] Zone Subset ACTSITE :HEM,56,184 Static monomer/residue 8

[1411] Color_Subset 255,255,0

[1412] Combine Subset ALLZONE Union ASP GLU

[1413] Combine Subset ALLZONE Union ALLZONE CTERM

[1414] Combine Subset ALLZONE Union ALLZONE ACTSITE

[1415] #NOTE: editnextline object name according to the protein

[1416] Combine Subset REST Difference ARP ALLZONE

[1417] List Subset REST Atom Output_File restatom. list

[1418] List Subset REST monomer/residue Output_File restmole.list

[1419] Color Molecule Atoms ACTSITE Specified Specification 255,0,0

[1420] List Subset ACTSITE Atom Output_File actsiteatom.list

[1421] List Subset ACTSITE monomer/residue output File

[1422] actsitemole.list

[1423] #

[1424] Zone Subset REST5A REST Static Monomer/Residue 5- Color_Subset

[1425] Combine Subset SUB5A Difference REST5A ACTSITE

[1426] Combine Subset SUB5B Difference SUB5A REST

[1427] Color Molecule Atoms SUB5B Specified Specification 255,255,255

[1428] List Subset SUB5B Atom Output_File sub5batom.list

[1429] List Subset SUB5B monomer/residue Output_File sub5bmole.list

[1430] #Now identify sites for asn→asp & gln→glu substitutions and . . .

[1431] #continue with makezone2.bcl.

[1432] #Use grep command to identify asn/gln in restatom.list

[1433] #sub5batom.list & accsiteatom.list

[1434] Comments:

[1435] The subset REST contains Gln70, and SUB5B contains Gln34, Asn128, Asn303 all of which are solvent exposed. The substitutions Q34E or Q34D, Q70E or Q70D, N128D or N128E and N303D or N303E are identified in A. ramosusperoxidase as sites for mutagenesis. Residues are substituted below and further analysis done:

[1436] Non-conservative Substitutions:

[1437] makeDEzone2.bcl

[1438] #sourcefile makezone2.bcl Claus von der Osten 961128

[1439] #

[1440] #having scanned lists (grep gln/asn command) and identified sites for . . .

[1441] #asn→asp & gln→glu substitutions

[1442] #NOTE: editnextline object name according to protein

[1443] Copy Object -To_Clipboard -Displace ARP newmodel Biopolymer

[1444] #NOTE: editnextline object name according to protein Blank Object On ARP

[1445] #NOTE: editnextlines with asn→asp & gln→glu positions

[1446] Replace Residue newmodel:34 glu L

[1447] Replace Residue newmodel:70 glu L

[1448] Replace Residue newmodel:128 asp L

[1449] Replace Residue newmodel:303 asp L

[1450] #Now repeat analysis done prior to asn→asp & gln→glu, . . .

[1451] #now including introduced asp & glu

[1452] Color Molecule Atoms newmodel Specified Specification 255,0,255

[1453] Zone Subset ASPx newmodel:asp:od* Static monomer/residue 10

[1454] Color_Subset 255,255,0

[1455] Zone Subset GLUx newmodel:glu:oe* Static monomer/residue 10

[1456] Color_Subset 255,255,0

[1457] #NOTE: editnextline C-terminal residue number according to the protein

[1458] Zone Subset CTERMX newmodel:344:0 Static monomer/residue 10

[1459] Color_Subset 255,255,0

[1460] #NOTE: editnextline ACTSITEx residues according to the protein

[1461] Zone Subset ACTSITEx newmodel:HEM,56,184 Static monomer/residue

[1462] 8 Color_Subset 255,255,0

[1463] Combine Subset ALLZONEx Union ASPx GLUx

[1464] Combine Subset ALLZONEx Union ALLZONEx CTERMx

[1465] Combine Subset ALLZONEx Union ALLZONEx ACTSITEx

[1466] Combine Subset RESTX Difference newmodel ALLZONEx

[1467] List Subset RESTx Atom Output_File restxatom.list

[1468] List Subset RESTX monomer/residue Output_File restxmole.list

[1469] #

[1470] Color Molecule Atoms ACTSITEx Specified Specification 255,0,0

[1471] List Subset ACTSITEx Atom Output_File actsitexatom.list

[1472] List Subset ACTSITEx monomer/residue Output_File

[1473] actsitexmole.list

[1474] #

[1475] #read restxatom.list or restxmole.list to identify sites for (not_gluasp)→gluasp . . .

[1476] #subst. if needed

[1477] Comments:

[1478] The subset RESTX contains only four residues: S9, S334, G335 and P336, all of which are >5% solvent exposed. The mutations S9D, S9E, S334D, S334E, G335D, G335E, P336D and P336E are proposed in A. ramosusperoxidase. Acidic residues within the subset ACTSITE are: E44, D57, D77, E87, E176, D179, E190, D202, D209, D246 and the N-terminal carboxylic acid on P344. Of these only E44, D77, E176, D179, E190, D209, D246 and the N-terminal carboxylic acid on P344 are solvent exposed. Suitable sites for mutations are E44Q, D77N, E176Q, D179N, E190Q, D209N and D246N. D246N and D246E are risky mutations due to D246's importance for binding of heme.

[1479] The N-terminal 8 residues were not included in the calculations above, as they do not appear in the structure. None of these 8 residues, QGPGGGG, contain carboxylic groups. The following variants are proposed as possible mutations to enable attachment to this region: Q1E, Q1, G2E, G2D, P3E, P3D, 45 G4E, G4D, G5E, G5D, G6E, G6D, G7E, G7D, G8E, G8D.

[1480] Relevant Data for Example 4:

[1481] Solvent accessibility data for A. ramosusperoxidase (Note: as the first eight residues are missing in the X-ray structure, the residue numbers printed in the accessibility list below are 8 lower than those used elsewhere for residue numbering.

[1482] Subset REST:

[1483] restmole.list

[1484] Subset REST:

[1485] ARP:9,69-70,125,127,133,299-301,334-336

[1486] restatom.list

[1487] Subset REST:

[1488] ARP:SER 9:N,CA,C,O,CB,OG

[1489] ARP:GLY 69:N,CA,C,O

[1490] ARP:GLN 70:N,CA,C,O,CB,CG,CD,OE1,NE2

[1491] ARP:GLY 125:N,CA,C,O

[1492] ARP:SER 127:N,CA,C,O,CB,OG

[1493] ARP:PRO 133:N,CA,CD,C,O,CB,CG

[1494] ARP:SER 299:N,CA,C,O,CB,OG

[1495] ARP:ALA 300:N,CA,C,O,CB

[1496] ARP:VAL 301:N,CA,C,O,CB,CG1,CG2

[1497] ARP:SER 334:N,CA,C,O,CB,OG

[1498] ARP:GLY 335:N,CA,C,O

[1499] ARP:PRO 336:N,CA,CD,C,O,CB,CG

[1500] Subset SUB5B:

[1501] sub5bmole.list

[1502] Subset SUB5B:

[1503] ARP:10-11,34,38,65-68,71-72,120-121,123-124,128-132,134,270,274,

[1504] ARP:297-298,302-303,311-312,332-333,337-338

[1505] sub5batom.list

[1506] Subset SUB5B:

[1507] ARP:VAL 10:N,CA,C,O,CB,CG1,CG2

[1508] ARP:THR 11:N,CA,C,O,CB,OG1,CG2

[1509] ARP:GLN 34:N,CA,C,O,CB,CG,CD,OE1,NE2

[1510] ARP:TYR 38:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1511] ARP:LEU 65:N,CA,C,O,CB,CG,CD1,CD2

[1512] ARP:THR 66:N,CA,C,O,CB,OG1,CG2

[1513] ARP:ALA 67:N,CA,C,O,CB

[1514] ARP:ALA 68:N,CA,C,O,CB

[1515] ARP:PHE 71:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1516] ARP:GLY 72:N,CA,C,O

[1517] ARP:PHE 120:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1518] ARP:ALA 121:N,CA,C,O,CB

[1519] ARP:ALA 123:N,CA,C,O,CB

[1520] ARP:VAL 124:N,CA,C,O,CB,CG1,CG2

[1521] ARP:ASN 128:N,CA,C,O,CB,CG,OD1,ND2

[1522] ARP:CYS 129:N,CA,C,O,CB,SG

[1523] ARP:PRO 130:N,CA,CD,C,O,CB,CG

[1524] ARP:GLY 131:N,CA,C,O

[1525] ARP:SER 132:N,CA,C,O,CB,OG

[1526] ARP:ARG 134:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1527] ARP:GLY 270:N,CA,C,O

[1528] ARP:ARG 274:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1529] ARP:ILE 297:N,CA,C,O,CB,CG1,CG2,CD1

[1530] ARP:PRO 298:N,CA,CD,C,O,CB,CG

[1531] ARP:SER 302:N,CA,C,O,CB,OG

[1532] ARP:ASN 303:N,CA,C,O,CB,CG,OD1,ND2

[1533] ARP:GLY 311:N,CA,C,O

[1534] ARP:GLY 312:N,CA,C,O

[1535] ARP:THR 332:N,CA,C,O,CB,OG1,CG2

[1536] ARP:ALA 333:N,CA,C,O,CB

[1537] ARP:LEU 337:N,CA,C,O,CB,CG,CD1,CD2

[1538] ARP:PRO 338:N,CA,CD,C,O,CB,CG

[1539] Subset ACTSITE:

[1540] actsitemole.list

[1541] Subset ACTSITE:

[1542] ARP:44-61,75-77,79-80,87-88,90-96,99,118,122,126,135,148-149,152-158,

[1543] ARP:163-164,167,176-194,197-205,207-209,211-213,216,230-231,241,

[1544] ARP:243-246,249,259,273,277,280,343-347H

[1545] actsiteatom.list

[1546] Subset ACTSITE:

[1547] ARP:GLU 44:N,CA,C,O,CB,CG,CD,OE1,OE2

[1548] ARP:SER 45:N,CA,C,O,CB,OG

[1549] ARP:PRO 46:N,CA,CD,C,O,CB,CG

[1550] ARP:VAL 47:N,CA,C,O,CB,CG1,CG2

[1551] ARP:ARG 48:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1552] ARP:LYS 49:N,CA,C,O,CB,CG,CD,CE,NZ

[1553] ARP:ILE 50:N,CA,C,O,CB,CG1,CG2,CD1

[1554] ARP:LEU 51:N,CA,C,O,CB,CG,CD1,CD2

[1555] ARP:ARG 52:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1556] ARP:ILE 53:N,CA,C,O,CB,CG1,CG2,CD1

[1557] ARP:VAL 54:N,CA,C,O,CB,CG1,CG2

[1558] ARP:PHE 55:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1559] ARP:HIS 56:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1560] ARP:ASP 57:N,CA,C,O,CB,CG,OD1,OD2

[1561] ARP:ALA 58:N,CA,C,O,CB

[1562] ARP:ILE 59:N,CA,C,O,CB,CG1,CG2,CD1

[1563] ARP:GLY 60:N,CA,C,O

[1564] ARP:PHE 61:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1565] ARP:GLY 15:N,CA,C,O

[1566] ARP:ALA 76:N,CA,C,O,CB

[1567] ARP:ASP 77:N,CA,C,O,CB,CG,OD1,OD2

[1568] ARP:SER 79:N,CA,C,O,CB,OG

[1569] ARP:ILE 80:N,CA,C,O,CB,CG1,CG2,CD1

[1570] ARP:GLU 87:N,CA,C,O,CB,CG,CD,OE1,OE2

[1571] ARP:LEU 88:N,CA,C,O,CB,CG,CD1,CD2

[1572] ARP:PHE 90:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1573] ARP:PRO 91:N,CA,CD,C,O,CB,CG

[1574] ARP:ALA 92:N,CA,C,O,CB

[1575] ARP:ASN 93:N,CA,C,O,CB,CG,OD1,ND2

[1576] ARP:GLY 94:N,CA,C,O

[1577] ARP:GLY 95:N,CA,C,O

[1578] ARP:LEU 96:N,CA,C,O,CB,CG,CD1,CD2

[1579] ARP:THR 99:N,CA,C,O,CB,OG1,CG2

[1580] ARP:ILE 118:N,CA,C,O,CB,CG1,CG2,CD1

[1581] ARP:THR 122:N,CA,C,O,CB,OG1,CG2

[1582] ARP:MET 126:N,CA,C,O,CB,CG,SD,CE

[1583] ARP:LEU 135:N,CA,C,O,CB,CG,CD1,CD2

[1584] ARP:SER 148:N,CA,C,O,CB,OG

[1585] ARP:PRO 149:N,CA,CD,C,O,CB,CG

[1586] ARP:LEU 152:N,CA,C,O,CB,CG,CD1,CD2

[1587] ARP:ILE 153:N,CA,C,O,CB,CG1,CG2,CD1

[1588] ARP:PRO 154:N,CA,CD,C,O,CB,CG

[1589] ARP:GLY 155:N,CA,C,O

[1590] ARP:PRO 156:N,CA,CD,C,O,CB,CG

[1591] ARP:GLY 157:N,CA,C,O

[1592] ARP:ASN 158:N,CA,C,O,CB,CG,OD1,ND2

[1593] ARP:ILE 163:N,CA,C,O,CB,CG1,CG2,CD1

[1594] ARP:LEU 164:N,CA,C,O,CB,CG,CD1,CD2

[1595] ARP:MET 167:N,CA,C,O,CB,CG,SD,CE

[1596] ARP:GLU 176:N,CA,C,O,CB,CG,CD,OE1,OE2

[1597] ARP:VAL 177:N,CA,C,O,CB,CG1,CG2

[1598] ARP:VAL 178:N,CA,C,O,CB,CG1,CG2

[1599] ARP:ASP 179:N,CA,C,O,CB,CG,OD1,OD2

[1600] ARP:LEU 180:N,CA,C,O,CB,CG,CD1,CD2

[1601] ARP:LEU 181:N,CA,C,O,CB,CG,CD1,CD2

[1602] ARP:ALA 182:N,CA,C,O,CB

[1603] ARP:ALA 183:N,CA,C,O,CB

[1604] ARP:HIS 184:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1605] ARP:SER 185:N,CA,C,O,CB,OG

[1606] ARP:LEU 186:N,CA,C,O,CB,CG,CD1,CD2

[1607] ARP:ALA 187:N,CA,C,O,CB

[1608] ARP:SER 188:N,CA,C,O,CB,OG

[1609] ARP:GLN 189:N,CA,C,O,CB,CG,CD,OE1,NE2

[1610] ARP:GLU 190:N,CA,C,O,CB,CG,CD,OE1,OE2

[1611] ARP:GLY 191:N,CA,C,O

[1612] ARP:LEU 192:N,CA,C,O,CB,CG,CD1, CD2

[1613] ARP:ASN 193:N,CA,C,O,CB,CG,OD1,ND2

[1614] ARP:SER 194:N,CA,C,O,CB,OG

[1615] ARP:PHE 197:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1616] ARP:ARG 198:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1617] ARP:SER 199:N,CA,C,O,CB,OG

[1618] ARP:PRO 200:N,CA,CD,C,O,CB,CG

[1619] ARP:LEU 201:N,CA,C,O,CB,CG,CD1,CD2

[1620] ARP:ASP 202:N,CA,C,O,CB,CG,OD1,OD2

[1621] ARP:SER 203:N,CA,C,O,CB,OG

[1622] ARP:THR 204:N,CA,C,O,CB,OG1,CG2

[1623] ARP:PRO 205:N,CA,CD,C,O,CB,CG

[1624] ARP:VAL 207:N,CA,C,O,CB,CG1,CG2

[1625] ARP:PHE 208:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1626] ARP:ASP 209:N,CA,C,O,CB,CG,OD1,OD2

[1627] ARP:GLN 211:N,CA,C,O,CB,CG,CD,OE1,NE2

[1628] ARP:PHE 212:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1629] ARP:TYR 213:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1630] ARP:THR 216:N,CA,C,O,CB,OG1,CG2

[1631] ARP:PHE 230:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1632] ARP:ALA 231:N,CA,C,O,CB

[1633] ARP:PHE241:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1634] ARP:MET 243:N,CA,C,O,CB,CG,SD,CE

[1635] ARP:ARG 244:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1636] ARP:SER 245:N,CA,C,O,CB,OG

[1637] ARP:ASP 246:N,CA,C,O,CB,CG,OD1,OD2

[1638] ARP:LEU 249:N,CA,C,O,CB,CG,CD1,CD2

[1639] ARP:TRP 259:N,CA,C,O,CB,CG,CD1,

[1640] CD2,NE1,CE2,CE3,CZ2,CZ3,CH2

[1641] ARP:TYR 273:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1642] ARP:MET 277:N,CA,C,O,CB,CG,SD,CE

[1643] ARP:MET 280:N,CA,C,O,CB,CG,SD,CE

[1644] ARP:ALA 343:N,CA,C,O,CB

[1645] ARP:PRO 344:N,CA,CD,C,O,OXT,CB,CG

[1646] ARP:HEM 345H:FE,NA,NB,NC,ND,CHA,CHB, CHC,CHD,C1A,C2A,C3A,C4A,CMA,CAA,CBA,CGA

[1647] ARP:HEM 345H:O1A,O2A,C1B,C2B,C3B,C4B,CMB, CAB,CBB,C1C,C2C,C3C,C4C,CMC,CAC,CBC

[1648] ARP:HEM 345H:C1D,C2D,C3D,C4D,CMD,CAD,CBD,CGD,O1D,O2D

[1649] ARP:CA 346H:CA

[1650] ARP:CA 347H:CA

[1651] Subset RESTx:

[1652] restxmole.list

[1653] Subset RESTX

[1654] NEWMODEL:9,334-336

[1655] restxatom.list

[1656] Subset RESTX:

[1657] NEWMODEL:SER 9:N,CA,C,O,CB,OG

[1658] NEWMODEL:SER 334:N,CA,C,O,CB,OG

[1659] NEWMODEL:GLY 335:N,CA,C,O

[1660] NEWMODEL:PRO 336:N,CA,CD,C,O,CB,CG

[1661] Activation of mPEG 15,000 With N-succinimidyl Carbonate

[1662] mPEG 15,000 was suspended in toluene (4 ml/g of mPEG) 20% was distilled off at normal pressure to dry the reactants azeotropically. Dichloromethane (dry 1 ml/g mPEG) was added when the solution was cooled to 30° C. and phosgene in toluene (1.93 M 5 mole/mole mPEG) was added and mixture stirred at room temperature overnight. The mixture was evaporated to dryness and the desired product was obtained as waxy lumps.

[1663] After evaporation dichloromethane and toluene (1:2, dry 3 ml/g mPEG) was added to re-dissolve the white solid. N-Hydroxy succinimide (2 mole/mole mPEG.) was added as a solid and then triethylamine (1.1 mole/mole mPEG). The mixture was stirred for 3 hours. initially unclear, then clear and ending with a small precipitate. The mixture was evaporated to dryness and recrystallized from ethyl acetate (10ml) with warm filtration to remove salts and insoluble traces. The blank liquid was left for slow cooling at ambient temperature for 16 hours and then in the refrigerator, overnight. The white precipitate was filtered and washed with a little cold ethyl acetate and dried to yield 98% (w/w). NMR Indicating 80-90% activation and 5 o/oo (w/w) HNEt₃Cl. ¹H-NMR for mPEG 15,000 (CDCl₃) d 1.42 t (I=4.8 CH₃i HNEt₃Cl), 2.84 s (I=3.7 succinimide), 3.10 dq (I=3.4 CH₂i HNEt₃Cl), 3.38 s (I=2.7 CH₃i OMe), 3.40* dd (I=4.5 o/oo, ¹³C satellite), 3.64 bs (I=1364 main peak), 3.89* dd (I=4.8 o/oo, ¹³C satellite), 4.47 dd (I=1.8, CH2 in PEG). No change was seen after storage in a desiccator at 22° C. for 4 months.

[1664] Activation of mPEG 5,000 With N-succinimidyl Carbonate

[1665] Activation of mPEG 5,000 with N-succinimidyl carbonate was performed as described in Example 5.

[1666] Construction and Expression of PD498 Variants:

[1667] PD498 site-directed variants were constructed using the “maxioligonucleotide-PCR” method described by Sarkar et al., (1990): BioTechniques 8: 404-407.

[1668] The template plasmid was shuttle vector pPD498 or an analogue of this containing a variant of the PD498 protease gene.

[1669] The following PD498 variants were constructed, expressed and purified.

[1670] A: R28K

[1671] B: R62K

[1672] C: R169K

[1673] D: R28K+R62K

[1674] E: R28K+R169K

[1675] F: R62K+R169K

[1676] G: R28K+R69K+R169K

[1677] Construction of Variants

[1678] For introduction of the R28K substitution a synthetic oligonucleotide having the sequence: GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO. 7) was used.

[1679] A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by StyI digestion and verified by DNA sequencing of the total 769 bp insert.

[1680] For introduction of the R62K substitution a synthetic oligonucleotide having the sequence: CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO. 8) was used.

[1681] A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by ClaI digestion and verified by DNA sequencing of the total 769 bp insert.

[1682] For introduction of the R169K substitution a synthetic oligonucleotide having the sequence: CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO. 9) was used.

[1683] A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by.Bst E II and Bgl II digestion. Positive variants were recognized by the absence of a Rsa I restriction site and verified by DNA sequencing of the total 769 bp insert.

[1684] For simultaneous introduction of the R28K and the R62K substitutions, synthetic oligonucleotides having the sequence: GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO. 7) and the sequence: CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID No. 8) were used simultaneous. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by StyI and ClaI digestion and verified by DNA sequencing of the total 769 bp insert.

[1685] For simultaneous introduction of the R28K and the R169K substitutions, synthetic oligonucleotides having the sequence: GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID NO. 7) and the sequence: CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO. 9) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by StyI digestion and absence of a Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.

[1686] For simultaneous introduction of the R62K and the R169K substitutions, synthetic oligonucleotides having the sequence: CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO. 8) and the sequence: CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO. 9) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by ClaI digestion and absence of a Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.

[1687] For simultaneous introduction of the R28K, the R62K and the 169K substitutions, synthetic oligonucleotides having the sequence: GGG ATG TAA CCA AGG GAA GCA GCA CTC AAA CG (SEQ ID No. 7) the sequence: CGA CTT TAT CGA TAA GGA CAA TAA CCC (SEQ ID NO. 8) and the sequence: CAA TGT ATC CAA AAC GTT CCA ACC AGC (SEQ ID NO. 9) were used simultaneously. A PCR fragment of 769 bp was ligated into the pPD498 plasmid prepared by Bst E II and Bgl II digestion. Positive variants were recognized by StyI and ClaI digestion and absence of a Rsa I site. The variant was verified by DNA sequencing of the total 769 bp insert.

[1688] Fermentation, Expression and Purification of PD498 Variants

[1689] Vectors hosting the above mentioned PD498 variants were purified from E. colicultures and transformed into B. subtilisin which organism the variants were fermented, expressed and purified as described in the “Materials and Methods” section above.

[1690] Conjugation of Triple Substituted PD498 Variant With Activated mPEG 5,000

[1691] 200 mg of triple substituted PD498 variant (i.e. the R28K+R62K+R169K substituted variant) was incubated in 50 mm NaBorate, pH 10, with 1.8 g of activated mPEG 5,000 with N-succinimidyl carbonate (prepared according to Example 2), in a final volume of 20 ml. The reaction was carried out at ambient temperature using magnetic stirring. Reaction time was 1 hour. The reaction was stopped by adding DMG buffer to a final concentration of 5 mM dimethyl glutarate, 1 mM CaCl₂and 50 mM borate, pH 5.0.

[1692] The molecule weight of the obtained derivative was approximately 120 kDa, corresponding to about 16 moles of mPEG attached per mole enzyme.

[1693] Compared to the parent enzyme, residual activity was close to 100% towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide).

[1694] Allergenicity Trails of PD498 Variant-SPEG5.000 in Guinea Pigs

[1695] Dunkin Hartley guinea pigs are stimulated with 1.0 μg PD498-SPEG 5,000 and 1.0 μg modified variant PD498-SPEG 5,000 by intratracheal installation.

[1696] Sera from immunized Dunkin Hartley guinea pigs are tested during the trial period in a specific IgG, ELISA (described above) to elucidate whether the molecules could activate the immune response system giving rise to a specific IgG₁response indicating an allergenic response.

[1697] The IgG₁levels of Dunkin Hartley guinea pigs during the trail period of 10 weeks are observed.

[1698] Suitable Substitutions in Humicola lanuginosaLipase for Addition of Amino Attachment Groups (—NH₂)

[1699] The 3D structure of Humicola lanuginosalipase (SEQ ID NO 6) is available in Brookhaven Databank as 1tib.pdb. The lipase consists of 269 amino acids.

[1700] The procedure described in Example 1 was followed. The sequence of H. lanuginosalipase is shown below in the table listing solvent accessibility data for H. lanuginosalipase. H. lanuginosaresidue numbering is used (1-269), and the active site residues (functional site) are S146, S201 and H258. The synonym TIB is used for H. lanuginosalipase.

[1701] The commands performed in Insight (BIOSYM) are shown in the command files makeKzone.bcl and makeKzone2.bcl below:

[1702] Conservative Substitutions:

[1703] makeKzone.bcl

[1704] 1 Delete Subset *

[1705] 2 Color Molecule Atoms * Specified Specification 255,0,255

[1706] 3 Zone Subset LYS :lys:NZ Static monomer/residue 10 Color_Subset 255,255,0

[1707] 4 Zone Subset NTERM :1:N Static monomer/residue 10 Color_Subset 255,255,0

[1708] 5 #NOTE: editnextline ACTSITE residues according to the protein

[1709] 6 Zone Subset ACTSITE :146,201,258 Static monomer/residue 8 Color_Subset 255,255,0

[1710] 7 Combine Subset ALLZONE Union LYS NTERM

[1711] 8 Combine Subset ALLZONE Union ALLZONE ACTSITE

[1712] 9 #NOTE: editnextline object name according to the protein

[1713] 10 Combine Subset REST Difference TIB ALLZONE

[1714] 11 List Subset REST Atom Output_File restatom.list

[1715] 12 List Subset REST monomer/residue Output_File restmole.list

[1716] 13 Color Molecule Atoms ACTSITE Specified Specification 255,0,0

[1717] 14 List Subset ACTSITE Atom Output_File actsiteatom.list

[1718] 15 List Subset ACTSITE monomer/residue.Output_File actsitemole.list

[1719] 16 #

[1720] 17 Zone Subset REST5A REST Static Monomer/Residue 5—Color_Subset

[1721] 18 Combine Subset SUB5A Difference REST5A ACTSITE

[1722] 19 Combine Subset SUB5B Difference SUB5A REST

[1723] 20 Color Molecule Atoms SUB5B Specified Specification 255,255,255

[1724] 21 List Subset SUB5B Atom Output_File sub5batom.list

[1725] 22 List Subset SUB5B monomer/residue Output_File sub5bmole.list

[1726] 23 #Now identify sites for lys→arg substitutions and continue with makezone2.bcl

[1727] 24 #Use grep command to identify ARG in restatom.list, sub5batom.list & accsiteatom.list

[1728] Comments:

[1729] In this case of H. lanuginosa(=TIB), REST contains the Arginines Arg133, Arg139, Arg160, Arg179 and Arg 209, and SUB5B contains Arg118 and R125.

[1730] These residues are all solvent exposed. The substitutions R133K, R139K, R160K, R179K, R209K, R118K and R125K are identified in TIB as sites for mutagenesis within the scope of this invention. The residues are substituted below in section 2, and further analysis done. The subset ACTSITE contains no lysines.

[1731] Non-conservative Substitutions:

[1732] makeKzone2.bcl

[1733] 1 #sourcefile makezone2.bcl Claus-von der Osten 961128

[1734] 2 #

[1735] 3 #having scanned lists (grep arg command) and identified sites for lys→arg substitutions

[1736] 4 #NOTE: editnextline object name according to protein

[1737] 5 Copy Object -To_Clipboard -Displace TIB newmodel

[1738] 6 Biopolymer

[1739] 7 #NOTE: editnextline object name according to protein

[1740] 8 Blank Object On TIB

[1741] 9 #NOTE: editnextlines with lys→arg positions

[1742] 10 Replace Residue newmodel:118 lys L

[1743] 11 Replace Residue newmodel:125 lys L

[1744] 12 Replace Residue newmodel:133 lys L

[1745] 13 Replace Residue newmodel:139 lys L

[1746] 14 Replace Residue newmodel:160 lys L

[1747] 15 Replace Residue newmodel:179 lys L

[1748] 16 Replace Residue newmodel:209 lys L

[1749] 17 #

[1750] 18 #Now repeat analysis done prior to arg→lys, now including introduced lysines

[1751] 19 Color Molecule Atoms newmodel Specified Specification 255,0,255

[1752] 20 Zone Subset LYSx newmodel:lys:NZ Static monomer/residue 10 Color_Subset 255,255,0

[1753] 21 Zone Subset NTERMx newmodel:1:N Static monomer/residue 10 Color_Subset 255,255,0

[1754] 22 #NOTE: editnextline ACTSITEx residues according to the protein

[1755] 23 Zone Subset ACTSITEx newmodel:146,201,258 Static monomer/residue 8 Color_Subset 255,255,0

[1756] 24 Combine Subset ALLZONEx Union LYSx NTERMx

[1757] 25 Combine Subset ALLZONEx Union ALLZONEx ACTSITEx

[1758] 26 Combine Subset RESTX Difference newmodel ALLZONEx

[1759] 27 List Subset RESTX Atom Output_File restxatom.list

[1760] 28 List Subset RESTX monomer/residue Output_File restxmole.list

[1761] 29 #

[1762] 30 Color Molecule Atoms ACTSITEx Specified Specification 255,0,0

[1763] 31 List Subset ACTSITEx Atom Output_File actsitexatom.list

[1764] 32 List Subset ACTSITEx monomer/residue Output_File actsitexmole.list

[1765] 33 #

[1766] 34 #read restxatom.list or restxmole.list to identify sites for (not_arg)→lys subst. if needed

[1767] Comments:

[1768] Of the residues in RESTX, the following are >5% exposed (see lists below): 18,31-33,36,38,40,48,50,56-62,64,78,88,91-93,104-106,120,136,225,227-229,250,262,268. Of these three are Cysteines involved in disulfide bridge formation, and consequently for structural reasons excluded from the residues to be mutated. The following mutations are proposed in H. lanuginosalipase (TIB): A18K,G31K,T32K,N33K,G38K,A40K,D48K,T50K,E56K,D57K,S58K,G59K, V60K,G61K,D62K,T64K,L78K,N88K,G91K,N92K,L93K,S105K,G106K, V120K,P136K,G225K,L227K,V228K,P229K,P250K,F262K. Relevant data for Example 2:

[1769] Subset REST:

[1770] restmole.list

[1771] Subset REST:

[1772] TIB:5,8-9,13-14,16,18-20,31-34,36,38,40,48-80,56-66,68,76-79,88,91-93,

[1773] TIB:100-107,116-117,119-121,132-134,136,139-142,154-40 169,177-185,

[1774] TIB:187,189-191,207-212,214-216,225,227-229,241-244,250,262,268

[1775] restatom.list

[1776] Subset REST:

[1777] TIB:ASP 5:N,CA,C,O,CB,CG,OD1,OD2

[1778] TIB:ASN 8:N,CA,C,O,CB,CG,OD1,ND2

[1779] TIB:GLN 9:N,CA,C,O,CB,CG,CD,OE1,NE2

[1780] TIB:PHE 13:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1781] TIB:ALA 14:N,CA,C,0,CB

[1782] TIB:TYR 16:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1783] TIB:ALA 18:N,CA,C,0,CB

[1784] TIB:ALA 19:N,CA,C,O,CB

[1785] TIB:ALA 20:N,CA,C,O,CB

[1786] TIB:GLY 31:N,CA,C,O

[1787] TIB:THR 32:N,CA,C,O,CB,OG1,CG2

[1788] TIB:ASN 33:N,CA,C,O,CB,CG,OD1,ND2

[1789] TIB:ILE 34:N,CA,C,O,CB,CG1,CG2,CD1

[1790] TIB:CYS 36:N,CA,C,O,CB,SG

[1791] TIB:GLY 38:N,CA,C,O

[1792] TIB:ALA 40:N,CA,C,O,CB

[1793] TIB:ASP 48:N,CA,C,O,CB,CG,OD1,OD2

[1794] TIB:ALA 49:N,CA,C,O,CB

[1795] TIB:THR 50:N,CA,C,O,CB,OG1,CG2

[1796] TIB:GLU 56:N,CA,C,O,CB,CG,CD,OE1,OE2

[1797] TIB:ASP 57:N,CA,C,O,CB,CG,OD1,OD2

[1798] TIB:SER 58:N,CA,C,O,CB,OG

[1799] TIB:GLY 59:N,CA,C,O

[1800] TIB:VAL 60:N,CA,C,O,CB,CG1,CG2

[1801] TIB:GLY 61:N,CA,C,O

[1802] TIB:ASP 62:N,CA,C,O,CB,CG,OD1,OD2

[1803] TIB:VAL 63:N,CA,C,O,CB,CG1,CG2

[1804] TIB:THR 64:N,CA,C,O,CB,OG1,CG2

[1805] TIB:GLY 65:N,CA,C,O

[1806] TIB:PHE 66:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1807] TIB:ALA 68:N,CA,C,O,CB

[1808] TIB:ILE 76:N,CA,C,O,CB,CG1,CG2,CD1

[1809] TIB:VAL 77:N,CA,C,O,CB,CG1,CG2

[1810] TIB:LEU 78:N,CA,C,O,CB,CG,CD1,CD2

[1811] TIB:SER 79:N,CA,C,O,CB,OG

[1812] TIB:ASN 88:N,CA,C,O,CB,CG,OD1,ND2

[1813] TIB:GLY 91:N,CA,C,O

[1814] TIB:ASN 92:N,CA,C,O,CB,CG,OD1,ND2

[1815] TIB:LEU 93:N,CA,C,O,CB,CG,CD1,CD2

[1816] TIB:ILE 100:N,CA,C,O,CB,CG1,CG2,CD1

[1817] TIB:ASN 101:N,CA,C,O,CB,CG,OD1,ND2

[1818] TIB:ASP 102:N,CA,C,O,CB,CG,OD1,OD2

[1819] TIB:ILE 103:N,CA,C,O,CB,CG1,CG2,CD1

[1820] TIB:CYS 104:N,CA,C,O,CB,SG

[1821] TIB:SER 105:N,CA,C,O,CB,OG

[1822] TIB:GLY 106:N,CA,C,O

[1823] TIB:CYS 107:N,CA,C,O,CB,SG

[1824] TIB:SER 116:N,CA,C,O,CB,OG

[1825] TIB:TRP 117:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2, CE3,CZ2,CZ3,CH2

[1826] TIB:SER 119:N,CA,C,O,CB,OG

[1827] TIB:VAL 120:N,CA,C,O,CB,CG1,CG2

[1828] TIB:ALA 121:N,CA,C,O,CB

[1829] TIB:VAL 132:N,CA,C,O,CB,CG1,CG2

[1830] TIB:ARG 133:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1831] TIB:GLU 134:N,CA,C,O,CB,CG,CD,OE1,OE2

[1832] TIB:PRO 136:N,CA,CD,C,O,CB,CG

[1833] TIB:ARG 139:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1834] TIB:VAL 140:N,CA,C.,O,CB,CG1,CG2

[1835] TIB:VAL 141:N,CA,C,O,CB,CG1,CG2

[1836] TIB:PHE 142:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1837] TIB:VAL 154:N,CA,C,O,CB,CG1,CG2

[1838] TIB:ALA 155:N,CA,C,O,CB

[1839] TIB:GLY 156:N,CA,C,O

[1840] TIB:ALA 157:N,CA,C,O,CB

[1841] TIB:ASP 158:N,CA,C,O,CB,CG,OD1,OD2

[1842] TIB:LEU 159:N,CA,C,O,CB,CG,CD1,CD2

[1843] TIB:ARG 160:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1844] TIB:GLY 161:N,CA,C,O

[1845] TIB:ASN 162:N,CA,C,O,CB,CG,OD1,ND2

[1846] TIB:GLY 163:N,CA,C,O

[1847] TIB:TYR 164:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1848] TIB:ASP 165:N,CA,C,O,CB,CG,OD1,OD2

[1849] TIB:ILE 166:N,CA,C,O,CB,CG1,CG2,CD1

[1850] TIB:ASP 167:N,CA,C,O,CB,CG,OD1,OD2

[1851] TIB:VAL 168:N,CA,C,O,CB,CG1,CG2

[1852] TIB:PHE 169:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1853] TIB:GLY 177:N,CA,C,O

[1854] TIB:ASN 178:N,CA,C,O,CB,CG,OD1,ND2

[1855] TIB:ARG 179:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1856] TIB:ALA 180:N,CA,C,O,CB

[1857] TIB:PHE 181:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1858] TIB:ALA 182:N,CA,C,O,CB

[1859] TIB:GLU 183:N,CA,C,O,CB,CG,CD,OE1,OE2

[1860] TIB:PHE 184:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1861] TIB:LEU 185:N,CA,C,O,CB,CG,CD1,CD2

[1862] TIB:VAL 187:N,CA,C,O,CB,CG1,CG2

[1863] TIB:THR 189:N,CA,C,O,CB,OG1,CG2

[1864] TIB:GLY 190:N,CA,C,O

[1865] TIB:GLY 191:N,CA,C,O

[1866] TIB:PRO 207:N,CA,CD,C,O,CB,CG

[1867] TIB:PRO 208:N,CA,CD,C,O,CB,CG

[1868] TIB:ARG 209:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1869] TIB:GLU 210:N,CA,C,O,CB,CG,CD,OE1,OE2

[1870] TIB:PHE 211:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1871] TIB:GLY 212:N,CA,C,O

[1872] TIB:SER 214:N,CA,C,O,CB,OG

[1873] TIB:HIS 215:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1874] TIB:SER 216:N,CA,C,O,CB,OG

[1875] TIB:GLY 225:N,CA,C,O

[1876] TIB:LEU 227:N,CA,C,O,CB,CG,CD1,CD2

[1877] TIB:VAL 228:N,CA,C,O,CB,CG1,CG2

[1878] TIB:PRO 229:N,CA,CD,C,O,CB,CG

[1879] TIB:ILE 241:N,CA,C,O,CB,CG1,CG2,CD1

[1880] TIB:ASP 242:N,CA,C,O,CB,CG,OD1,OD2

[1881] TIB:ALA 243:N,CA,C,O,CB

[1882] TIB:THR 244:N,CA,C,O,CB,OG1,CG2

[1883] TIB:PRO 250:N,CA,CD,C,O,CB,CG

[1884] TIB:PHE 262:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1885] TIB:CYS 268:N,CA,C,O,CB,SG

[1886] Subset SUB5B:

[1887] sub5mole.list

[1888] Subset SUB5B:

[1889] TIB:3-4,6-7,10-12,15,22-23,25-30,35,37,39,41-42,44-47,51-55,67,69-70,

[1890] TIB:72,74-75,94-99,108-112,114-115,118,122-126,128-131,135,137-138,

[1891] TIB:186,188,192-195,213,217-219,223-224,230-231,234-235,238-240,

[1892] TIB:245,269

[1893] sub5batom.list

[1894] Subset SUB5B:

[1895] TIB:SER 3:N,CA,C,O,CB,OG

[1896] TIB:GLN 4:N,CA,C,O,CB,CG,CD,OE1,NE2

[1897] TIB:LEU 6:N,CA,C,O,CB,CG,CD1,CD2

[1898] TIB:PHE 7:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1899] TIB:PHE 10:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1900] TIB:ASN 11:N,CA,C,O,CB,CG,OD1,ND2

[1901] TIB.:LEU 12:N,CA,C,O,CB,CG,CD1,CD2

[1902] TIB:GLN 15:N,CA,C,O,CB,CG,CD,OE1,NE2

[1903] TIB:CYS 22:N,CA,C,O,CB,SG

[1904] TIB:GLY 23:N,CA,C,O

[1905] TIB:ASN 25:N,CA,C,O,CB,CG,OD1,ND2

[1906] TIB:ASN 26:N,CA,C,O,CB,CG,OD1,ND2

[1907] TIB:ASP 27:N,CA,C,0,CB,CG,OD1,OD2

[1908] TIB:ALA 28:N,CA,C,O,CB

[1909] TIB:PRO 29:N,CA,CD,C,O,CB,CG

[1910] TIB:ALA 30:N,CA,C,O,CB

[1911] TIB:THR 35:N,CA,C,O,CB,OG1,CG2

[1912] TIB:THR 37:N,CA,C,O,CB,OG1,CG2

[1913] TIB:ASN 39:N,CA,C,O,CB,CG,OD1,ND2

[1914] TIB:CYS 41:N,CA,C,O,CB,SG

[1915] TIB:PRO 42:N,CA,CD,C,O,CB,CG

[1916] TIB:VAL 44:N,CA,C,O,CB,CG1,CG2

[1917] TIB:GLU-45:N,CA,C,O,CB,CG,CD,OE1,OE2

[1918] TIB:LYS 46:N,CA,C,O,CB,CG,CD,CE,NZ

[1919] TIB:ALA 47:N,CA,C,O,CB

[1920] TIB:PHE 51:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1921] TIB:LEU 52:N,CA,C,O,CB,CG,CD1,CD2

[1922] TIB:TYR 53:N,CA,C,O;CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1923] TIB:SER 54:N,CA,C,O,CB,OG

[1924] TIB:PHE 55:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1925] TIB:LEU 67:N,CA,C,O,CB,CG,CD1,CD2

[1926] TIB:LEU 69:N,CA,C,O,CB,CG,CD1,CD2

[1927] TIB:ASP 70:N,CA,C,O,CB,CG,OD1,OD2

[1928] TIB:THR 72:N,CA,C,O,CB,OG1,CG2

[1929] TIB:LYS 74:N,CA,C,O,CB,CG,CD,CE,NZ

[1930] TIB:LEU 75:N,CA,C,O,CB,CG,CD1,CD2

[1931] TIB:ASN 94:N,CA,C,O,CB,CG,OD1,ND2

[1932] TIB:PHE 95:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1933] TIB:ASP 96:N,CA,C,O,CB,CG,OD1,OD2

[1934] TIB:LEU 97:N,CA,C,O,CB,CG,CD1,CD2

[1935] TIB:LYS 98:N,CA,C,O,CB,CG,CD,CE,NZ

[1936] TIB:GLU 99:N,CA,C,O,CB,CG,CD,OE1,OE2

[1937] TIB:ARG 108:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1938] TIB:GLY 109:N,CA,C,O

[1939] TIB:HIS 110:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1940] TIB:ASP 111:N,CA,C,O,CB,CG,OD1,OD2

[1941] TIB:GLY 112:N,CA,C,O

[1942] TIB:THR 114:N,CA,C,O,CB,OG1,CG2

[1943] TIB:SER 115:N,CA,C,O,CB,OG

[1944] TIB:ARG 118:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1945] TIB:ASP 122:N,CA,C,O,CB,CG,OD1,OD2

[1946] TIB:THR 123:N,CA,C,O,CB,OG1,CG2

[1947] TIB:LEU 124:N,CA,C,O,CB,CG,CD1,CD2

[1948] TIB:ARG 125:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1949] TIB:GLN 126:N,CA,C,O,CB,CG,CD,OE1,NE2

[1950] TIB:VAL 128:N,CA,C,O,CB,CG1,CG2

[1951] TIB:GLU 129:N,CA,C,O,CB,CG,CD,OE1,OE2

[1952] TIB:ASP 130:N,CA,C,O,CB,CG,OD1,OD2

[1953] TIB:ALA 131:N,CA,C,O,CB

[1954] TIB:HIS 135:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[1955] TIB:ASP 137:N,CA,C,O,CB,CG,OD1,OD2

[1956] TIB:TYR 138:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1957] TIB:THR 186:N,CA,C,O,CB,OG1,CG2

[1958] TIB:GLN 188:N,CA,C,O,CB,CG,CD,OE1,NE2

[1959] TIB:THR 192:N,CA,C,O,CB,OG1,CG2

[1960] TIB:LEU 193:N,CA,C,O,CB,CG,CD1,CD2

[1961] TIB:TYR 194:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1962] TIB:ARG 195:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1963] TIB:TYR 213:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1964] TIB:SER 217:N,CA,C,O,CB,OG

[1965] TIB:PRO 218:N,CA,CD,C,O,CB,CG

[1966] TIB:GLU 219:N,CA,C,O,CB,CG,CD,OE1,OE2

[1967] TIB:LYS 223:N,CA,C,O,CB,CG,CD,CE,NZ

[1968] TIB:SER 224:N,CA,C,O,CB,OG

[1969] TIB:VAL 230:N,CA,C,O,CB,CG1,CG2

[1970] TIB:THR 231:N,CA,C,O,CB,OG1,CG2

[1971] TIB:ASP 234:N,CA,C,O,CB,CG,OD1,OD2

[1972] TIB:ILE 235:N,CA,C,O,CB,CG1,CG2,CD1

[1973] TIB:ILE 238:N,CA,C,O,CB,CG1,CG2,CD1

[1974] TIB:GLU 239:N,CA,C, 0,CB,CG,CD,OE1,OE2

[1975] TIB:GLY 240:N,CA,C,O

[1976] TIB:GLY 245:N,CA,C,O

[1977] TIB:LEU 269:N,CA,C,O,CB,OXT,CG,CD1,CD2

[1978] Subset ACTSITE:

[1979] actsitemole.list

[1980] Subset ACTSITE:

[1981] TIB:17,21,80-87,89-90,113,143-153,170-176,196-206,221-222,226,246-249,

[1982] TIB:251-261,263-267

[1983] actsiteatom.list

[1984] Subset ACTSITE:

[1985] TIB:SER 17:N,CA,C,0,CB,OG

[1986] TIB:TYR 21:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[1987] TIB:PHE 80:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1988] TIB:ARG 81:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1989] TIB:GLY 82:N,CA,C,O

[1990] TIB:SER 83:N,CA,C,O,CB,OG

[1991] TIB:ARG 84:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[1992] TIB:SER 85:N,CA,C,O,CB,OG

[1993] TIB:ILE 86:N,CA,C,O,CB,CG1,CG2,CD1

[1994] TIB:GLU 87:N,CA,C,O,CB,CG,CD,OE1,OE2

[1995] TIB:TRP 89:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2

[1996] TIB:ILE 90:N,CA,C,O,CB,CG1,CG2,CD1

[1997] TIB:PHE 113:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[1998] TIB:THR 143:N,CA,C,O,CB,OG1,CG2

[1999] TIB:GLY 144:N,CA,C,O

[2000] TIB:HIS 145:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[2001] TIB:SER 146:N,CA,C,O,CB,OG

[2002] TIB:LEU 147:N,CA,C,O,CB,CG,CD1,CD2

[2003] TIB:GLY 148:N,CA,C,O

[2004] TIB:GLY 149:N,CA,C,O

[2005] TIB:ALA 150:N,CA,C,O,CB

[2006] TIB:LEU 151:N,CA,C,O,CB,CG,CD1,CD2

[2007] TIB:ALA 152:N,CA,C,O,CB

[2008] TIB:THR 153:N,CA,C,O,CB,OG1,CG2

[2009] TIB:SER 170:N,CA,C,O,CB,OG

[2010] TIB:TYR 171:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[2011] TIB:GLY 172:N,CA,C,O

[2012] TIB:ALA 173:N,CA,C,O,CB

[2013] TIB:PRO 174:N,CA,CD,C,O,CB,CG

[2014] TIB:ARG 175:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[2015] TIB:VAL 176:N,CA,C,O,CB,CG1,CG2

[2016] TIB:ILE 196:N,CA,C,O,CB,CG1,CG2,CD1

[2017] TIB:THR 197:N,CA,C,O,CB,OG1,CG2

[2018] TIB:HIS 198:N,CA,C,00CB,CG,ND1,CD2,CE1,NE2

[2019] TIB:THR 199:N,CA,C,O,CB,OG1,CG2

[2020] TIB:ASN 200:N,CA,C,O,CB,CG,OD1,ND2

[2021] TIB:ASP 201:N,CA,C,O,CB,CG,OD1,OD2

[2022] TIB:ILE 202:N,CA,C,O,CB,CG1,CG2,CD1

[2023] TIB:VAL 203:N,CA,C,O,CB,CG1,CG2

[2024] TIB:PRO 204:N,CA,CD,C,O,CB,CG

[2025] TIB:ARG 205:N,CA,C,O,CB,CG,CD,NE,CZ,NH1,NH2

[2026] TIB:LEU 206:N,CA,C,O,CB,CG,CD1,CD2

[2027] TIB:TRP 221:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2

[2028] TIB:ILE 222:N,CA,C,O,CB,CG1,CG2,CD1

[2029] TIB:THR 226:N,CA,C,O,CB,OG1,CG2

[2030] TIB:GLY 246:N,CA,C,O

[2031] TIB:ASN 247:N,CA,C,O,CB,CG,OD1,ND2

[2032] TIB:ASN 248:N,CA,C,O,CB,CG,OD1,ND2

[2033] TIB:GLN 249:N,CA,C,O,CB,CG,CD,OE1,NE2

[2034] TIB:ASN 251:N,CA,C,O,CB,CG,OD1,ND2

[2035] TIB:ILE 252:N,CA,C,O,CB,CG1,CG2,CD1

[2036] TIB:PRO 253:N,CA,CD,C,O,CB,CG

[2037] TIB:ASP 254:N,CA,C,O,CB,CG,OD1,OD2

[2038] TIB:ILE 255:N,CA,C,O,CB,CG1,CG2,CD1

[2039] TIB:PRO 256:N,CA,CD,C,O,CB,CG

[2040] TIB:ALA 257:N,CA,C,O,CB

[2041] TIB:HIS 258:N,CA,C,O,CB,CG,ND1,CD2,CE1,NE2

[2042] TIB:LEU 259:N,CA,C,O,CB,CG,CD1,CD2

[2043] TIB:TRP 260:N,CA,C,O,CB,CG,CD1,CD2,NE1,CE2,CE3,CZ2,CZ3,CH2

[2044] TIB:TYR 261:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[2045] TIB:GLY 263:N,CA,C,O

[2046] TIB:LEU 264:N,CA,C,O,CB,CG,CD1,CD2

[2047] TIB:ILE 265:N,CA,C,O,CB,CG1,CG2,CD1

[2048] TIB:GLY 266:N,CA,C,O

[2049] TIB:THR 267:N,CA,C,O,CB,OG1,CG2

[2050] Subset RESTX:

[2051] restxmole.list

[2052] Subset RESTX:

[2053] NEWMODEL:14,16,18-20,31-34,36,38,40,48-50,56-66,68,78-79,88,91-93,

[2054] NEWMODEL:104-106,120,136,225,227-229,250,262,268

[2055] restxatom.list

[2056] Subset RESTX:

[2057] NEWMODEL:ALA 14:N,CA,C,O,CB

[2058] NEWMODEL:TYR 16:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ,OH

[2059] NEWMODEL:ALA 18:N,CA,C,O,CB

[2060] NEWMODEL:ALA 19:N,CA,C,O,CB

[2061] NEWMODEL:ALA 20:N,CA,C,O,CB

[2062] NEWMODEL:GLY 31:N,CA,C,O

[2063] NEWMODEL:THR 32:N,CA,C,O,CB,OG1,CG2

[2064] NEWMODEL:ASN 33:N,CA,C,O,CB,CG,OD1,ND2

[2065] NEWMODEL:ILE 34:N,CA,C,O,CB,CG1,CG2,CD1

[2066] NEWMODEL:CYS 36:N,CA,C,O,CB,SG

[2067] NEWMODEL:GLY 38:N,CA,C,O

[2068] NEWMODEL:ALA 40:N,CA,C,O,CB

[2069] NEWMODEL:ASP 48:N,CA,C,O,CB,CG,OD1,OD2

[2070] NEWMODEL:ALA 49:N,CA,C,O,CB

[2071] NEWMODEL:THR 50:N,CA,C,O,CB,OG1,CG2

[2072] NEWMODEL:GLU 56:N,CA,C,O,CB,CG,CD,OE1,OE2

[2073] NEWMODEL:ASP 57:N,CA,C,O,CB,CG,OD1,OD2

[2074] NEWMODEL:SER 58:N,CA,C,O,CB,OG

[2075] NEWMODEL:GLY 59:N,CA,C,O

[2076] NEWMODEL:VAL 60:N,CA,C,O,CB,CG1,CG2

[2077] NEWMODEL:GLY 61:N,CA,C,O

[2078] NEWMODEL:ASP 62:N,CA,C,O,CB,CG,OD1,OD2

[2079] NEWMODEL:VAL 63:N,CA,C,O,CB,CG1,CG2

[2080] NEWMODEL:THR 64:N,CA,C,O,CB,OG1,CG2

[2081] NEWMODEL:GLY 65:N,CA,C,O

[2082] NEWMODEL:PHE 66:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[2083] NEWMODEL:ALA 68:N,CA,C,O,CB

[2084] NEWMODEL:LEU 78:N,CA,C,O,CB,CG,CD1,CD2

[2085] NEWMODEL:SER 79:N,CA,C,O,CB,OG

[2086] NEWMODEL:ASN 88:N,CA,C,O,CB,CG,OD1,ND2

[2087] NEWMODEL:GLY 91:N,CA,C,O

[2088] NEWMODEL:ASN 92:N,CA,C,O,CB,CG,OD1,ND2

[2089] NEWMODEL:LEU 93:N,CA,C,O,CB,CG,CD1,CD2

[2090] NEWMODEL:CYS 104:N,CA,C,O,CB,SG

[2091] NEWMODEL:SER 105:N,CA,C,O,CB,OG

[2092] NEWMODEL:GLY 106:N,CA,C,O

[2093] NEWMODEL:VAL 120:N,CA,C,O,CB,CG1,CG2

[2094] NEWMODEL:PRO 136:N,CA,CD,C,O,CB,CG

[2095] NEWMODEL:GLY 225:N,CA,C,O

[2096] NEWMODEL:LEU 227:N,CA,C,O,CB,CG,CD1,CD2

[2097] NEWMODEL:VAL 228:N,CA,C,O,CB,CG1,CG2

[2098] NEWMODEL:PRO 229:N,CA,CD,C,O,CB,CG

[2099] NEWMODEL:PRO 250:N,CA,CD,C,O,CB,CG

[2100] NEWMODEL:PHE 262:N,CA,C,O,CB,CG,CD1,CD2,CE1,CE2,CZ

[2101] NEWMODEL:CYS 268:N,CA,C,O,CB,SG

[2102] Providing a Lipase Variant E87K+D254K

[2103] The Humicola lanuginosalipase variant E87K+D254K was constructed, expressed and purified as described in WO 92/05249.

[2104] Lipase-S-PEG 15,000 Conjugate

[2105] The lipase variant E87K+D254K-SPEG conjugate was prepared as described in Example 7, except that the enzyme is the Humicola lanuginosalipase variant (E87K+D254K) described in Example 10 and the polymer is mPEG15,000.

[2106] Immunogenecity Assessed as IgG, of Lipase Variant (D87K+D254K) in Balb/C Mice

[2107] Balb/c mice were immunized by subcutanuous injection of:

[2108] i) 50 μl 0.9% (wt/vol) NaCl solution (control group, 8 mice) (control),

[2109] ii) 50 μl 0.9% (wt/vol) NaCl solution containing 25 μg of protein of a Humicola lanuginosalipase variant (E87K+D254K) (group 1, 8 mice) (unmodified lipase variant),

[2110] iii) 50% 0.9% (wt/vol) NaCl solution containing a Humicola lanugoinosalipase variant substituted in position D87K+D254K and coupled to a N-succinimidyl carbonate activated mPEG 15,000(group 2, 8 mice) (lipase-SPEG15,000).

[2111] The amount of protein for each batch was measured by optical density measurements. Blood samples (200 p1) were collected from the eyes one week after the immunization, but before the following immunization. Serum was obtained by blood clotting, and centrifugation.

[2112] The IgG₁response was determined by use of the Balb/C mice IgG, ELISA method as described above.

[2113] Results:

[2114] Five weekly immunizations were required to elicit a detectable humoral response to the unmodified Humicola lanuginosavariant. The antibody titers elicited by the conjugate (i.e. lipase-SPEG15,000 ranged between 960 and 1920, and were only 2 to 4× lower than the antibody titer of 3840 that was elicited by unmodified HL82-Lipolase (figure to the left).

[2115] The Results of the Tests are Shown in FIG. 1

[2116] As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.