Antiviral Protein

The present invention relates to a Mirabilis Antiviral Protein (to be referred to as MAP hereinafter) variant and, more particularly, to a MAP variant whose inhibition activity in in vitro protein synthesis is improved as compared with that of natural MAP.

2. Description of the Related Art

The present inventors previously separated a novel basic protein from Mirabilis jalapa and found that this protein exhibited antiviral activity against a plurality of plant viruses. This protein was called MAP and was claimed in Published Examined Japanese Patent Application No. 63-61317. All amino acid sequences, synthesis of a gene based on the specified amino acid sequences, and construction of a system in E. coli, in which MAP is produced and secreted into the medium using this complete or full synthetic MAP gene were claimed in Published Unexamined Japanese Patent Application No. 2-186988 and Japanese Patent Application No. 1-210767.

The MAP is a type of ribosome inactivating protein (RIP) widely existing in plants and microorganisms, and exhibits an RNA N-glycosidase activity with high specificity for ribosomal RNA as a substrate. Ribosomes are inactivated by this activity, inhibiting protein synthesis, as is well known. Such protein synthesis inhibition activity is highly toxic in cells. In recent years, this toxicity has been utilized to develop immunotoxins having high selectivity, for example, by linking a ricin A chain, a type of RIP derived from Ricinus communis, to various antibodies. These immunotoxins are utilized, for example, in missile therapy.

This protein toxin possesses high antigenicity. There exists the possibility that an antibody against the toxin is produced in living bodies. Thus, long-term doses may adversely affect the living body. Therefore, proteins possessing various properties for use as toxic proteins are required.

Under these circumstances, the MAP is one of the most promising candidates as a toxin for an immunotoxin. The protein synthesis inhibition activity in a rabbit reticulocyte system has been shown to be only about 1/30 of the ricin A chain.

Protein engineering involving gene manipulation techniques has developed remarkably in recent years. In various applications, amino acid sequences of natural proteins are altered to produce proteins whose inherent activities are modified. It has been found in these applications that disulfide bonds (S-S bonds) in protein molecules are closely associated with the flexibility of the protein molecule, and that the activity can be greatly changed, depending on the presence or absence of S-S bonds (Matsumura et. al., Nature 342, pp. 291-293 (1989); Matsumura et. al., Proc. Natl. Acad. Sci. USA 87, pp. 6562-6566 (1989); Kozo Hamaguchi, Biochemistry 1, pp. 1-13 (1991); and Pace, Biotrend 2-4, pp. 105-110 (1990)).

When the foregoing is taken into consideration, the production of proteins possessing greater activity and, more particularly, possessing greater protein synthesis inhibition activity is expected by utilizing MAP genes.

It is an object of the present invention to provide a protein having high protein synthesis inhibition activity and, more particularly, a protein having higher protein synthesis inhibition activity than that of MAP.

The inventors have conducted extensive studies to achieve the above object, and have discovered a MAP variant having a higher protein synthesis inhibition activity than that of MAP by changing two cysteine residues associated with an S-S bond of the MAP into serine residues. That is, the antiviral protein according to the present invention is a MAP variant having an amino acid sequence represented by SEQ ID No:8 in the Sequence Listing to be described later.

This MAP variant having no cysteine bond (to be referred to as MAP-H hereinafter) is produced by converting a codon encoding cysteine into a codon encoding serine in the MAP gene, inserting the resultant gene into a MAP secretion vector, and introducing this vector into a host such as E. coli.

The MAP gene is a gene encoding MAP. An amino acid and a codon specifying this amino acid are not generally set in a one-to-one correspondence. One to six types of codons specifying one amino acid are generally present. A large number of types of MAP genes are present, and a large number of types of recombinant genes in which codons encoding cysteines are substituted with codons encoding serines are accordingly present. When this recombinant gene is to be introduced into a host cell to produce an antiviral protein of the present invention, all the recombinant genes can be utilized. When a specific amino acid is taken into consideration, however, the frequency of use of several types of codons encoding specific amino acids can be unbalanced, depending on the species. Thus, when E. coli is the host, a gene having the base sequence represented by SEQ ID No:1 in the Sequence Listing is preferably employed.

Production of a MAP gene based on a MAP amino acid sequence is described in detail in Japanese Patent Application No. 63-93494. According to this method, a codon encoding cysteine is substituted with a codon encoding serine to obtain a total synthetic gene encoding the antiviral protein of the present invention. An E. coli transformant harboring a vector containing a total synthetic MAP gene produced by the above method has already been deposited in the Fermentation Research Institute (FRI) as deposit No. 9913. A total synthetic MAP gene can be extracted from this transformant, and only the codon encoding cysteine is substituted with a codon encoding serine to obtain a gene encoding the antiviral protein of the present invention.

Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate a presently preferred embodiment of the invention and, together with the general description given above and the detailed description of the preferred embodiment given below, serve to explain the principles of the invention.

FIG. 1 shows the base sequence of a synthetic gene encoding MAP;

FIG. 2 shows an OmpA signal sequence and the base sequence of the gene encoding the OmpA signal sequence, together with part of a MAP amino acid sequence and the base sequence of the gene encoding the MAP;

FIG. 3 shows the base sequence of a synthetic DNA linker;

FIG. 4 schematically illustrates step A in the Example of the present invention;

FIG. 5 schematically illustrates step B in the Example of the present invention;

FIG. 6 schematically illustrates step C in the Example of the present invention;

FIG. 7 schematically illustrates step E in the Example of the present invention;

FIG. 8 schematically illustrates steps F and G in the Example of the present invention;

FIG. 9 schematically illustrates steps H and I in the Example of the present invention;

FIG. 10 schematically illustrates steps J and K in the Example of the present invention;

FIG. 11 shows the amino acid sequence of block II inserted in a MAP variant and the base sequence of a synthetic DNA fragment encoding this amino acid sequence according to the Example of the present invention;

FIG. 12 shows the amino acid sequence of block VII inserted in a MAP variant and the base sequence of a synthetic DNA fragment encoding this amino acid sequence according to the Example of the present invention;

FIG. 13 is a photograph showing the migration pattern of a MAP variant by SDS polyacrylamide gel electrophoresis; and

FIG. 14 is a graph showing the protein synthesis inhibition activities of MAP and a MAP variant having no S-S bond in a rabbit reticulocyte system.

A method of preparing an antiviral protein according to the present invention will be described in detail wherein a total MAP gene extracted from a deposited E. coli transformant is used.

A total synthetic MAP gene (SEQ ID NO:1) contained in the deposited E. coli transformant is constructed by linking the eight blocks I to VIII shown in FIG. 1. The blocks are linked at different restriction sites, respectively. A desired block can be obtained by using a specific restriction enzyme. Codons encoding two cysteine residues present in the MAP amino acid sequence are respectively present in blocks II and VII of this total synthetic MAP gene. In order to obtain a MAP gene encoding the MAP variant of the present invention, DNA fragments of these portions are newly synthesized, substituting a codon encoding cysteine with a codon encoding serine, and then the MAP gene is reconstructed using the new fragments in place of the fragments of blocks II and VII in the units of blocks.

For this purpose, a DNA fragment substituted with a target codon is synthesized and integrated in a plasmid. The plasmid containing this DNA fragment is cloned. At this time, it is desirable that the plasmid have the same base sequence as the original sequence, except that a restriction enzyme site is additionally inserted or deleted, and that the plasmid is cloned by use of the site as a marker. Note that synthesis is performed by using a commercially available DNA synthesizer, and purification is performed using HPLC. Known methods are used for phenol treatment and ethanol precipitation necessary for treating DNA, DNA digestion or cleavage by restriction enzymes, subsequent DNA recovery, the calcium treatment serving as the E. coli transformation method using the plasmid DNA, the alkaline-SDS method serving as the method of purifying DNA from E. coli transformants, and the dideoxy method serving as the sequence determination method for nucleic acids. For cloning, a presynthesized DNA is annealed such that the terminal ends of the resultant double-stranded DNA have EcoRI and HindIII sites, and the resultant double-stranded DNA is easily inserted into the plasmid, at which the same sites as the terminal ends of the resultant double-stranded DNA are present.

Since the terminals of each block of the MAP gene form restriction enzyme sites, each cloned DNA fragment is cleaved at the corresponding site, and the cleaved fragment is then inserted into a total synthetic MAP gene. Insertion of each fragment is confirmed by the presence/absence of a newly inserted restriction enzyme site or a deleted restriction enzyme site. The inserted MAP gene is cleaved again with an appropriate restriction enzyme, and the cleaved portions are linked to each other to complete production of a MAP variant (i.e., MAP-H gene).

A vector for introducing a foreign gene which is to be expressed in E. coli generally requires the following DNA sequences other than the gene to be introduced:

(a) a region for controlling transcription (operator).

(b) a region for promoting initiation of gene transcription (promoter).

Examples of promoters which are known to function in E. coli are the N25 promoter and the P.sub.L promoter, both of which are derived from coliphage, etc. The P.sub.L promoter is derived from E. coli lambda-phage, and is known to be repressed by a control protein which is called cI. The cI includes a temperature-sensitive variant, called cI.sub.857, which represses the P.sub.L promoter at 30.degree. C., like the cI, but which loses its repressing ability at 42.degree. C., resulting in P.sub.L promoter activity. Accordingly, if an expression vector introduced in E. coli includes both the P.sub.L promoter and the cI.sub.857 repressor gene, E. coli can be grown by culturing it at 30.degree. C. such that the P.sub.L promoter is repressed. Also, the P.sub.L promoter can be turned on to initiate transcription of the gene by culturing E. coli at 42.degree. C.

The P.sub.L promoter can be obtained by digesting lambda-phage DNA or the disclosed pPL-lambda plasmid with restriction enzymes BamHI and HpaI. The cI.sub.887 repressor gene can be obtained by digesting DNA of lambda-phage variant (cI.sub.857, Sam 7) with the restriction enzymes BglII and BanIII.

(c) a region for controlling the termination of transcription (terminator).

Examples of known terminators are the tLI terminator derived from coliphage, the rrnBT.sub.1 T.sub.2 terminator derived from ribosome genes of E. coli, etc..

(d) a region for controlling the position of initiation of translation after transcription into mRNA (Shine-Dalgarno, SD sequence).

A sequence which is common to genes of E. coli can be used as an SD sequence.

(e) a methionine codon linked to the SD sequence for initiation of translation (ATG).

An expression vector derived from plasmid DNA can be constructed by deletion or insertion of a specific region from the plasmid DNA. The deletion and insertion can be performed by cleaving the plasmid at specific sites and combining the resultant fragments by means of an appropriate treatment. Specifically, appropriate utilization of synthetic DNA fragments enables restriction enzyme sites, an SD sequence, a gene encoding the amino acid sequence of a protein, etc. which are not present in the original plasmid DNA to be introduced.

For example, by making a DNA fragment including XbaI and BanIII sites that are not included in plasmid pKK223-3 and its complementary chain to combine with a cleavage fragment (a large fragment) which is obtained by digesting pKK223-3 with EcoRI and HindIII, the restriction enzyme sites for XbaI and BanIII can be introduced into pKK223-3. Additionally, by utilizing this pKK223-3 which includes the restriction enzyme sites XbaI and BanIII and a total synthetic MAP gene also having XbaI and BanIII sites at its 5'- and 3'-terminals, respectively, the total synthetic MAP gene can be introduced into plasmid pKK223-3. Further, an SD sequence and a codon encoding a methionine residue which is required for initiating gene transcription can also be introduced into said plasmid at this time. Moreover, since the restriction enzyme site of NdeI (CATATG) includes a methionine codon (ATG) therein, a gene encoding other proteins can be introduced through this site.

Required DNA fragments can be synthesized by a DNA synthesizer. An expression vector is formed by combining DNA fragments obtained from the DNA synthesizer, those obtained by restriction enzyme cleavage, and such a DNA fragment as needed, obtained by converting a cohesive end into a flush end utilizing T4 DNA polymerase, DNA polymerase Klenow fragment, etc. Each fragment can be combined using T4 DNA ligase or a commercially available ligation kit including the DNA ligase.

IV) Production of a MAP Secretion Vector

As described above, the total synthetic MAP gene having, e.g., the P.sub.L promotor and cI.sub.857 gene obtained from the E. coli transformant (deposit No. 9913) deposited in the FRI, is integrated in an expression vector, and the expression vector is introduced into E. coli, thereby expressing the gene and hence mass-producing MAP in E. coli. In this case, the mass-produced MAP is stored in E. coli. This MAP affects the growth of E. coli and, thus, the production of MAP is limited. The mass-produced protein must be secreted outside E. coli.

OmpA is an outer membrane protein of E. coli and comprises a signal sequence (SE ID No:2) as shown in FIG. 2. The base sequence of a gene encoding this signal sequence is also shown in FIG. 2. This signal sequence functions to make OmpA secrete from E. coli. Therefore, by linking this signal sequence to the N-terminal end of other proteins, these proteins can be transferred outside from the inside of E. coli. For example, by linking the signal sequence shown in FIG. 2 to the N-terminal end of MAP, MAP can be secreted from E. coli.

Further, the gene encoding the signal sequence has the first methionine codon included in a part of said NdeI site, and thus a foreign gene can be introduced into an expression vector having an NdeI site downstream from a promoter.

FIG. 2 also shows a sequence of three amino acids of the MAP N-terminal region, and a DNA sequence corresponding thereto.

The MAP secretion vector obtained in step IV has a gene containing the OmpA signal sequence, and the MAP gene is inserted downstream thereof. The cleavage site of restriction enzyme XbaI is present in the N-terminal sequence of the MAP gene, and the BanIII site is present downstream of the C-terminal end. Therefore, the MAP-H gene obtained in step II is inserted using these two sites.

If the MAP secretion vector used in step V has, e.g., the P.sub.L promotor and cI.sub.857 coding for its control protein, expression of the MAP gene located downstream of the promotor is completely repressed at 30.degree. C., and cI.sub.857 is inactivated at 42.degree. C. to immediately express the MAP gene, thereby producing the MAP. The signal sequence of E. coli outer membrane protein OmpA is inserted upstream of the MAP gene, so that the produced MAP is immediately secreted into the medium. A transformant containing the plasmid into which the MAP-H gene is inserted is cultured at a low temperature. When the concentration of the transformant reaches an appropriate value, the MAP-H gene is expressed by using the known temperature shift. The MAP-H is produced from E. coli, and is secreted into the medium. The resultant MAP-H is condensed as a precipitate by salting out with ammonium sulfate and then dialyzed. The dialyzed MAP-H is purified using Carboxymethyl Sepharose and Blue Sepharose column chromatography.

The MAP or MAP-H as a MAP variant is quantitatively evaluated by an ELISA (Enzyme-Linked Immunosorbent Assay) using an antiserum against MAP. In in vitro protein synthesis, i.e., translation, the RNA of tobacco mosaic virus is added as the mRNA in a protein synthesis system of a commercially available rabbit reticulocyte crude extract, and evaluation is performed using the content of labeled amino acid (.sup.35 S-methionine) present in the acid-insoluble polypeptide produced by the translation. At this time, an appropriate amount of MAP or MAP-H is added to the system to quantitatively evaluate its influence on this protein synthesis.

The present invention will be described in more detail by way of the following examples. The present invention, however, is not limited to the following examples. In order to readily understand the present invention, steps A, B, C, and E are illustrated in FIGS. 4, 5, 6, and 7, respectively; steps F and G are illustrated in FIG. 8; steps H and I are illustrated in FIG. 9; and steps J and K are illustrated in FIG. 10.

A DNA linker (SEQ ID No: 3) encoding restriction enzyme sites, an SD sequence, a methionine codon, and the N-terminal amino acid sequence of MAP shown in FIG. 3 was inserted into plasmid pKK223-3 extracted from E. coli (strain HB 101) transformed by a known method.

One microgram of pKK223-3 was incubated in High Salt Buffer (a mixture of 50 mM Tris-HCl, pH 7.5-100 mM NaCi-1 mM MgCl.sub.2) containing 10 units each of restriction enzymes EcoRI and HindIII (manufactured by Nippon Gene Co, Ltd.) at 37.degree. C. for one hour for digestion. The obtained solution was subjected to phenol-chloroform treatment and ethanol precipitation to collect the DNA. The phenol-chloroform treatment was as follows. Firstly, phenol was saturated with a mixture (hereinafter abbreviated as TE) of 10 mM Tris-HCl, pH 8.0, 1 mM ethylenediamine tetraacetic acid (EDTA). The equivalent volume of the resultant phenol solution was added to the obtained DNA solution for mixing, and the resultant mixture was centrifuged to collect the aqueous phase containing the DNA. Next, an equivalent volume of chloroform was added to this aqueous phase for further mixing, and the resultant mixture was centrifuged to collect the aqueous phase containing the DNA. Ethanol precipitation was performed as follows. Firstly, to the obtained solution containing the DNA, 5M sodium chloride, 1/20-fold volume, and ethanol, 2-fold volume, were added, and the resultant mixture was cooled at -70.degree. C. for thirty minutes. Next, this solution was centrifuged at high speed to recover the obtained precipitate.

Two kinds of single-stranded synthetic DNA linkers, having the base sequences shown in FIG. 3, complementary to each other, were prepared by utilizing a DNA synthesizer (manufactured by Applied Biosystems Japan Company, 381A-type) according to the phosphoramidide method. One microgram of each obtained synthetic linker was incubated in 100 .mu.l of a kinase solution (a mixture of 50 mM Tris-HCl, pH 7.6, 10 mM MgCl.sub.2, 5 mM dithiothreitol, 0.1 mM spermidine, 0.1 mM EDTA, and 1 mM ATP) containing 10 units of T4 kinase (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for one hour to add phosphoric acid to the 5'-terminal end of the linkers. After that, the obtained single-stranded DNA was converted into double-stranded DNA by annealing. This annealing was performed by mixing the obtained reacted solutions, heating the resultant mixture at 60.degree. C. for twenty minutes, and allowing it to stand at room temperature for twenty minutes. Next, the resultant solution was subjected to ethanol precipitation, and then the precipitate was dissolved in 10 .mu.l of TE.

To 5 .mu.l of the thus obtained solution containing double-stranded synthetic DNA, 2.5 .mu.l (ca. 0.5 .mu.g) of pKK223-3 cleavage product was added, and the mixture was ligated at 10.degree. C. for two hours utilizing a ligation kit (manufactured by Takara Shuzo Co., Ltd). After that, the resultant DNA was utilized to transform E. coli (strain HB101). From the obtained transformants, plasmid pKS2 was prepared. Plasmid pKS2 is the plasmid formed by inserting the synthetic DNA linker into plasmid pKK223-3.

Two micrograms of plasmid pKS2 and restriction enzyme BamHl were incubated at 37.degree. C. for one hour in 50 .mu.l of High Salt Buffer for digestion. Next, the obtained solution was subjected to phenol-chloroform treatment and ethanol precipitation to collect the cleaved DNA. The obtained precipitate was added to 25 .mu.l of Klenow solution (which is obtained by adding 0.1 mM of each co-factor dATP, dGTP, dCTP and TTP to a mixture of 50 mM Tris-HCl, pH 7.2, 10 mM MgSO.sub.4, 0.1 mM dithiothreitol, and 50 .mu.g/ml of bovine serum albumin) containing 2 units of Klenow fragment (manufactured by Toyobo Co., Ltd.), and the resultant solution was incubated at 22.degree. C. for 30 minutes to convert the cohesive ends of the DNA to flush ends. After the reaction, the solution was heated at 70.degree. C. for five minutes, followed by phenol-chloroform treatment and ethanol precipitation to collect the DNA. The collected DNA was further cleaved by dissolving it in 50 .mu.l of High Salt Buffer containing 10 units of restriction enzyme PvuI (manufactured by Toyobo Co., Ltd.) and incubating at 37.degree. C. for one hour. The cleaved DNA was collected by phenol-chloroform treatment, followed by ethanol precipitation.

Separately, 1 .mu.g of plasmid pUC 19 (manufactured by Takara Syuzo Co., Ltd.) was incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes PvuI (manufactured by Toyobo Co., Ltd.) and PvuII (manufactured by Nippon Gene Ltd.) at 37.degree. C. for two hours to cleave pUC19. The cleaved DNA fragments were collected by phenol-chloroform treatment, followed by ethanol precipitation.

The DNA fragment (larger fragment) derived from pKS2 and the DNA fragment derived from pUC19, both of which were obtained as described, were dissolved in 10 .mu.l of TE, respectively. After that, 3.5 .mu.l of each of the TE solutions were mixed, and the fragments were ligated at 10.degree. C. for one hour utilizing a ligation kit (manufactured by Takara Shuzo Co., Ltd.). After that, the resultant DNA was utilized to transform E. coli (strain HB101). From the obtained transformants, plasmid pKS3 was prepared. pKS3 comprises the replication origin of pUC19 and the large fragment of pKS2 which are combined therein.

Two micrograms of pKS3 were incubated in 50 .mu.l of High Salt Buffer containing 10 units of restriction enzyme PstI (manufactured by Nippon Gene Co., Ltd.) at 37.degree. C. for one hour for digestion. The cleaved DNA fragment was collected by phenol-chloroform treatment followed by ethanol precipitation. The collected DNA fragment was incubated in 20 .mu.l of polymerase solution (which was obtained by adding 0.1 mM of each co-factor dATP, dGTP, dCTP and TTP to a mixture of 33 mM Tris-HCl, pH 7.9, 66 mM potassium phosphate, 10 mM magnesium acetate, 0.5 mM dithiothreitol, and 0.1 mg/ml of bovine serum albumin) containing 2.5 units of T4 DNA polymerase (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for five minutes to convert the cohesive end of the DNA fragment to a flush end. Next, 1 .mu.l of 0.5M EDTA was added to the resultant solution, and the obtained mixture was subjected to phenol-chloroform treatment and further ethanol precipitation to collect the DNA. The collected DNA was incubated in 50 .mu.l of High Salt Buffer containing 10 units of restriction enzyme BamHl (manufactured by Nippon Gene Co., Ltd.) at 37.degree. C. for one hour for additional digestion.

Separately, 1 .mu.g of pPL-lambda (manufactured by Pharmacia Co., Ltd.) was incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes BamHI and HpaI (both manufactured by Nippon Gene Co., Ltd.) at 37.degree. C. for one hour for digestion. Next, the mixture was subjected to phenol-chloroform treatment and ethanol precipitation to collect the DNA fragment containing the P.sub.L promotor.

The DNA fragment derived from pKS3 and the DNA fragment containing the P.sub.L promoter were dissolved in 10 .mu.l of TE, respectively. Next, 3.5 .mu.l of each solution were mixed, and the DNA fragments therein were ligated utilizing a ligation kit (manufactured by Takara Syuzo Col, Ltd.). After that, the resultant DNA was utilized to transform E. coli (strain HB101). From the obtained transformants, plasmid pSH4 was prepared. pSH4 is the plasmid which was formed by inserting the P.sub.L promotor into pKS3.

Two micrograms of lambda-phage (lambda, cI.sub.857, Sam7) DNA (manufactured by Takara Syuzo Co., Ltd.) were incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes BglII (manufactured by Nippon Gene Co., Ltd.) and BanIII (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for two hours for digestion. The obtained DNA fragments were collected by phenolchloroform treatment followed by ethanol precipitation.

Meanwhile, 1 .mu.g of plasmid pHSG397 (manufactured by Takara Syuzo Col, Ltd.) was incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes BamHI (Nippon Gene Co., Ltd.) and BanIII (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for one hour for digestion. Next, to the mixture, 2 .mu.l (1 unit) of alkaline phosphatase (manufactured by Toyobo Co,, Ltd.) were added, and the resultant mixture was heated at 60.degree. C. for 30 minutes for dephosphorylation at the 5'-terminal end of the DNA. After that, the DNA was collected by phenol-chloroform treatment and ethanol precipitation.

The lambda-phage DNA cleavage product and the pHSG 397 cleavage product which were thus obtained were each dissolved in 10 .mu.l of TE. Next, 3.5 .mu.l of each solution were mixed and the DNA cleavage products therein were ligated by reacting the mixture at 10.degree. C. for two hours utilizing a ligation kit (manufactured by Takara Syuzo Co., Ltd.). The obtained DNA was utilized to transform E. coli (strain HB101), and plasmid DNA was purified from the obtained transformants. This plasmid DNA is pHSGcI.sub.857, formed by inserting the BglII-BanIII fragment of ca.1100 base pairs including cI.sub.857 into pHSG397.

E) The Step of Inserting cI.sub.857 into pSH4

Two micrograms of pHSGcI.sub.857 were incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes XhoI (manufactured by Nippon Gene Co., Ltd.) and BanIII (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for one hour for digestion. The cleaved DNA fragments were collected by phenol-chloroform treatment and ethanol precipitation. The collected DNA was incubated in 25 .mu.l of Klenow solution containing two units of Klenow fragment at 22.degree. C. for 30 minutes to convert the cohesive end of the DNA to a flush end. Next, the resultant solution was heated at 70.degree. C. for five minutes and subjected to phenol-chloroform treatment and ethanol precipitation to collect the DNA.

On the other hand, 1 .mu.g of pSH4 was incubated in 50 .mu.l of High Salt Buffer containing 10 units of restriction enzyme BamH1 (manufactured by Nippon Gene Co., Ltd.) at 37.degree. C. for one hour for digestion. The cleaved DNA was collected by phenol-chloroform treatment and ethanol precipitation. The collected DNA was incubated in 25 .mu.l of Klenow solution containing 2 units of Klenow fragment at 22.degree. C. for 30 minutes to convert the cohesive end of the DNA to a flush end. Next, the resultant solution was heated at 70.degree. C. for five minutes, and subjected to phenol-chloroform treatment and ethanol precipitation to collect the resultant DNA.

The DNA fragment including cI.sub.857 and the cleaved pSH4 fragments which were thus obtained were dissolved in 10 .mu.l of TE, respectively. Next, 3.5 .mu.l of each solution were mixed and the DNA fragments were ligated by reacting the solution at 10.degree. C. for two hours utilizing a ligation kit (manufactured by Takara Syuzo Co., Ltd.). The obtained DNA was utilized to transform E. coli (strain HB101), and plasmid DNA was purified from the obtained transformants. The obtained plasmid is pSH5 formed by inserting cI.sub.857 into plasmid pSH4.

Two micrograms of pSH5 were incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes XbaI (manufactured by Nippon Gene Co., Ltd.) and BanIII (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for one hour for digestion The resultant solution was subjected to phenol-chloroform treatment and ethanol precipitation to collect the cleaved DNA.

Meanwhile, 2 .mu.g of pMHI were digested and the cleaved DNA was collected in the same manner as for pSHS. pMHI is a synthetic plasmid formed by inserting a total synthetic MAP gene into the plasmid pUC19 and extracting it from the above-mentioned E. coli transformant (deposit No. 9913) deposited in the FIR.

The DNA fragment derived from pSH5 and the fragment from pMHI thus obtained were each dissolved in 10 .mu.l of TE, respectively. Next, 3.5 .mu.l of each solution were mixed and the DNA fragments therein were ligated by reacting the resultant mixture at 10.degree. C. for one hour utilizing a ligation kit (manufactured by Takara Syuzo Co., Ltd.). The combined DNA was used to transform E. coli (strain N99cI+), and plasmid DNA was purified from the obtained transformants. This plasmid is pSH6 formed by inserting a fragment of the total synthetic MAP gene, which was obtained by cleaving with XbaI and BanIII, into the plasmid pHS5.

Each single-stranded DNA of complementary DNA fragments having the base sequences shown in FIG. 2 was synthesized according to the phosphoramidide method utilizing a DNA synthesizer (manufactured by Applied Biosystems Japan, type 381A). One microgram of each synthesized single-stranded DNA was incubated in 50 .mu.l of said kinase solution containing 10 units of T4 kinase (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for one hour to phosphorylate the 5'-terminal end of the DNA. Each solution containing the phosphorylated single-stranded DNA was mixed, and the resultant solution was heated at 60.degree. C. for 20 minutes and allowed to stand at room temperature for 20 minutes for annealing to obtain double-stranded DNA. The obtained double-stranded DNA was collected by ethanol precipitation and dissolved in 10 .mu.l of TE.

On the other hand, 1 .mu.g of the plasmid pSH6 was incubated in 50 .mu.l of High Salt Buffer containing 10 units each of restriction enzymes NdeI and XbaI (both manufactured by Nippon Gene Co., Ltd.) at 37.degree. C. for one hour for digestion. The cleaved DNA was collected by subjecting the reacted solution to phenol-chloroform treatment and ethanol precipitation. The collected DNA was further dissolved in 10 .mu.l of TE.

3.5 .mu.l of the TE solution containing the annealed synthetic DNA and 3.5 .mu.l of the TE solution containing the cleaved pSH6 were mixed. The mixture was reacted at 10.degree. C. for two hours by utilizing a ligation kit manufactured by Takara Syuzo Co., Ltd.) to combine the synthetic DNA and the cleaved DNA. The combined DNA was used to transform E. coli (strain N99cI+), and plasmid DNA was purified from the obtained transformants. This plasmid is plasmid pSH7 formed by inserting the OmpA signal sequence gene into pSH6.

DNA fragments having the base sequence (SEQ ID NO: 4) shown in FIG. 11 as four fragments and DNA fragments having the base sequence (SEQ ID NO: 6) shown in FIG. 12 as two fragments were synthesized by a DNA synthesizer (manufactured by Applied Biosystems Japan Company, type 381A), respectively. The synthesized products were DNA fragments of blocks II and VII of MAP-H genes. In this case, codons encoding serines in place of those encoding cysteines were used, being "TCG" and "TCT" respectively However, the codons are not limited to the above. Any codon may be used if it encodes serine.

One microgram of each fragment was incubated in 50 .mu.l of a kinase solution containing 10 units of T4 kinase (manufactured by Toyobo Co., Ltd.) at 37.degree. C. for one hour to phosphorylate its 5'-terminal end. The four kinds of incubated solutions, each containing different fragments, were mixed for block II, and the two kinds of incubated solutions, each containing different fragments, were mixed for block VII at 60.degree. C. for 20 minutes, and the resultant solutions were left to stand for an hour at room temperature, thereby annealing complementary chains. Five .mu.l of each resultant solution were added to 2.5 .mu.l of a solution containing 0.1 .mu.g of DNA obtained by cleaving plasmid pUC 19 with the restriction enzymes EcoRI and HindIII, treating with phenol and precipitating with ethanol, and were linked using a commercially available ligation kit (manufactured by Takara Shuzo Co., Ltd.). E. coli strain HB101 was transformed by the calcium method using the resultant plasmid, and the plasmid was purified from the resultant transformant by the alkaline-SDS method, thereby confirming that the synthesized DNA fragments were inserted. This confirmation was performed by DNA sequence determination according to the dideoxy method. Plasmid pUCMBC36S was obtained by insertion of fragments of block II, and plasmid pUCMBC220S was obtained by insertion of fragments of block VII.

Block II was cleaved out from the resultant plasmid pUCMBC36S by the restriction enzymes SacI and SplI, and block VII was cleaved out from the resultant plasmid pUCMBC220S by the restriction enzymes Eco521 and BstEII. The cleaved blocks are inserted into the corresponding blocks of the complete synthetic MAP gene and thus substitute for them as follows. About 1 .mu.g of each DNA cleaved by the corresponding restriction enzymes was subjected to phenol treatment and ethanol precipitation and was dissolved in 10 .mu.l of a solution containing 10 mM Tris-HCl, pH 8.0 and 10 mM EDTA, respectively. 3.5 .mu.l of each resultant solution were mixed, and the DNAs contained in each solution were ligated to each other using a ligation kit (manufactured by Takara Shuzo Co., Ltd.). The complete MAP gene was extracted from the E. coli transformant deposited in the FRI as deposit No. 9913. Using the resultant plasmid, i.e., a plasmid obtained by inserting a small fragment (i.e., block II or VII) cleaved out from the plasmid pUCMBC36S or pUCMBC220S into the complete synthetic MAP gene (pMH1), E. coli (strain HB101) was transformed by the calcium method. The plasmid was purified by the alkaline-SDS method from the resultant transformant. Insertion of each block was confirmed by the presence of a newly introduced SalI site for block II and by the absence of a deleted PvuI site for block VII. Plasmid pUCMAPC36S was obtained upon insertion of block II, and plasmid pUCMAPC220S was obtained upon insertion of block VII.

The plasmids pUCMAPC36S and pUCMAPC220S were cleaved by restriction enzymes SacI and SpII. The block II obtained by cleaving plasmid pUCMAPC36S was further cleaved by restriction enzyme ScaI so as not to convert it into the original plasmid. In a sample containing block VII obtained by cleaving plasmid pUCMAPC220S, alkaline phosphatase (manufactured by Toyobo Co., Ltd.) was added to the reacted solution to eliminate phosphoric acid at its 5'terminal end, so that the resultant DNA was not converted into the original plasmid unless other DNAs were inserted at positions cleaved by the restriction enzymes.

The respective plasmid cleavage products were subjected to phenol treatment and ethanol precipitation. Each sample was then dissolved in 10 .mu.l of a solution containing 10 mM Tris-HCl, pH 8.0 and 10 mM EDTA, and 3.5 .mu.l each of the resultant solutions were mixed to link them using a ligation kit (manufactured by Takara Shuzo Co., Ltd.) E. coli (strain MVl184) was transformed using the resultant plasmid in which both blocks II and VII were inserted. The plasmid was purified by the alkaline-SDS method from the resultant transformant. Insertion was confirmed by the presence/absence of the restriction enzyme site as in the previous step, thereby obtaining plasmid pUCMAP-Ho This plasmid contains the MAP-H gene having a base sequence represented as SEQ ID NO:1 in the Sequence Listing.

The plasmid pUCMAP-H obtained in step J and the MAP expression secretion vector pSH7 obtained in step G were cleaved with restriction enzymes XbaI and BanIII, respectively, and were subjected to phenol treatment and ethanol precipitation. Each sample was dissolved in 10 .mu.l of a solution containing 10 mM Tris-HCl, pH 8.0 and 10 mM EDTA, and 3.5 .mu.l of each of the resultant solutions were mixed and linked using a ligation kit (manufactured by Takara Shuzo Co., Ltd.). E. coli (strain 99ci+) was transformed by the calcium method using the resultant plasmid. The plasmid was then purified from the resultant transformant by the alkaline-SDS method, thereby obtaining plasmid pSH7H, in which the MAP-H gene was inserted in pSH7. This E. coli transformant harboring plasmid pSH7H has been deposited in the FRI (deposit No.: FRI No. 12093).

E. coli (strain MM294) was transformed by the calcium method using secretion vector pSH7H in which the MAP-H gene was inserted. The resultant transformant was subjected to shaking culture in 2l of L medium (a mixture of 1% bact.trypton, 0.5% bact.yeast extract, 0.5% sodium chloride, and 0.1% glucose) at 30.degree. C. When the absorption of the medium at 550 nm reached 0.8, an equivalent amount of medium preheated to 55.degree. C. was added to the above medium, and the temperature of the total medium was kept at 42.degree. C. The shaking culture was continued at this temperature for 3 hours. Thereafter, ammonium sulfate was added to the medium from which bacteria had been eliminated by centrifugation, so that the concentration of ammonium sulfate was 90% to saturation, and the protein was salted out. This protein was precipitated by centrifugation and was collected. The collected protein was dissolved in 40 ml of A buffer solution (10 mM sodium phosphate buffer solution, pH 6.0), and the A buffer solution was dialyzed. The resultant crude extract was chromatographed on a Carboxymethyl Sepharose column (26 mm in diameter and 40 mm in length) pretreated with the A buffer solution. After the column was sufficiently washed with the A buffer solution, the adsorbed protein was eluted with a sodium chloride solution having a linear gradient of 0M to 0.5M. The MAP-H fractions of the eluted protein were identified and collected by ELISA using an anti-MAP antiserum. The collected proteins were dialyzed using a B buffer solution (10 mM Tris-HCl, pH 8.0) and were chromatographed on a Blue Sepharose column (5 mm in diameter and 50 mm in length) pretreated with B buffer solution. After the column was sufficiently washed with the B buffer solution, the protein was eluted with a sodium chloride solution containing a linear gradient of 0M to 0.2M. MAP-H fractions of the eluted protein were identified and collected by ELISA using an anti-MAP antiserum, and were dialyzed using distilled water, thereby purifying MAP-H. This MAP-H was analyzed by SDS-polyacrylamide gel electrophoresis, and the result is shown in FIG. 13. Referring to FIG. 13, lane 1 represents molecular weight markers, i.e., each band represents 97, 66, 42, 30, and 20 kilodaltons from the top, respectively. Lanes 2 to 4 represent natural MAPs, and lane 5 represents the MAP-H of the present invention. The MAP in lane 2 was reduced with 2-mercaptoethanol immediately before electrophoresis to cleave S-S bonds. As compared with the MAP in lane 4, which was not subjected to reduction, the pattern is found to be changed (a and b). The MAP-H in lane 5 shows the same pattern as the MAP in which the S-S bond is cleaved. Almost no bands representing other impurities are found in the MAP-H migration pattern shown in FIG. 13, and the protein is found to be almost uniform.

Tobacco mosaic virus RNA as mRNA was added to 10 .mu.l of a commercially available rabbit reticulocyte crude extract containing .sup.35 S-methionine and an appropriate amount of MAP or MAP-H, and the resultant mixture was incubated for translation at 30.degree. C. for 30 minutes. Two .mu.l of this reacted solution were placed on filter paper and dried, and this paper was boiled in a 10% trichloroacetic acid solution for 10 minutes. The radioactivity of the radioactive material (polypeptide containing incorporated .sup.35 S) left on the filter paper was measured using a toluene-based scintillator. The radioactivity of a sample not containing the mRNA was defined as 0%, the radioactivity of a sample not containing the MAP or MAP-H was defined as 100% as a control, and the effect of the MAP and MAP-H were determined. The results are shown in FIG. 14. Referring to FIG. 14, the ratio of the amount of incorporated .sup.35 S compared to that of the control is plotted along the ordinate, and the MAP or MAP-H concentration is plotted along the abscissa.

As is apparent from FIG. 14, MAP at about 3.5 mM exhibited a 50% inhibition effect in this system, and MAP-H at about 0.16 nM exhibited a 50% inhibition effect. Thus, the protein synthesis inhibition activity of MAP-H was improved about 22 times compared to that of the natural MAP.

As has bee described above in detail, the MAP variant (MAP-H) having no S-S bond and serving as an antiviral protein according to the present invention exhibits a higher protein synthesis inhibition activity than that of the natural protein, while preserving the advantages of the natural protein. Therefore, the antiviral protein according to the present invention is most promising as a toxic protein used as, e.g., an immunotoxin.

Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and illustrated examples shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.