Tracheal Antimicrobial Peptides, DNA Sequences and Methods for the Production and Use Thereof

The present invention relates generally to antimicrobial peptides referred to herein as tracheal antimicrobial peptides. More particularly, the present invention is related to a new class of polypeptides with antimicrobial activity, cDNA sequences encoding for the peptides, methods for the production and use thereof.

The respiratory epithelium of mammals is a complex tissue responsible for numerous physiological functions, one of which is forming a key barrier to potentially harmful environmental threats. Multiple defense mechanisms have been identified which protect the respiratory tract from inhaled agents that are known to be responsible for airway disease, such as infectious agents, gases, and particulates. Newhouse, M.T. and J. Bienenstock, "Respiratory Tract Defense Mechanisms," Textbook of Pulmonary Disease, Little, Brown and Comp (1989). These multiple defenses are the result of a combination of anatomical design of the airway, together with the physiological role of local and circulating cells.

Recent isolation and characterization of antimicrobial peptides in a variety of species and tissues has unveiled a new component of animal host defense. These various peptides, which can be classified into families based on common sequences, secondary structure and/or sites of activity, are believed to participate in defense against potential microbiological pathogens. Cecropins were the first well characterized family of structurally related antimicrobial peptides and are found in a wide distribution of insects. Boman, H.G. and D. Hultmark, Ann. Rev. Microbiol., 41:103-126 (1987). They are coordinately expressed in the fat body of insect larvae following infection or injury. In vertebrates, the magainin family of antimicrobial peptides have been isolated from glands of the skin and gastrointestinal tract of Xenopus Iaevis, and are thought to form the basis for a defense system of the amphibian mucosal surfaces against infection. Soravia, E., G. Martini et al., "Antimicrobial properties of peptides from Xenopus granular gland secretions," FEBS Lett., 228:337-40 (1988); Zasloff, M.A., "Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active froma, and partial cDNA sequence of a precursor," Proc Natl Acad Sci USA, 84:5449-53 (1987). Defensins are peptides found in phagocytic cells isolated from several mammalian species including man, and may be characterized by 8 invariant residues within the sequence. Gabay, J.E., "Microbicidal mechanisms of phagocytes," Curr Opin Immunol, 1(1):36-40 (1988); Gabay, J.E., R.W. Scott et al., "Antibiotic proteins of human polymorphonuclear leukocytes," Proc Natl Acad Sci USA, 86(14):5610-4 (1989); Ganz, T., "Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes," Infect Immun, 55(3):568-71 (1987): Ganz, T., J.A. Metcalf et al., "Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi syndrome and `specific` granule deficiency," J CIin Invest, 82(2):552-6 (1988); Ganz, T., J.R. Rayner et al., "The structure of the rabbit macrophage defensin genes and their organ-specific expression," J Immunol, 143(4):1358-65 (1989); Ganz, T., M.E. Selsted et al., "Antimicrobial activity of phagocyte granule proteins," Semin Respir Infect, 1(2):107-17 (1986); Ganz, T., M.E. Selsted et al., "Defensins," Eur J Hematol, 44(1):1-8 (1990a); Ganz, T., M.E. Selsted et al., "Defensins," Eur J Haematol, 44(1):1-8 (1990b); Ganz, T., M.E. Selsted et al., "Defensins. Natural peptide antibodies of human neutrophils," J Clin Invest, 76(4):1427-35 (1985). They possess antimicrobial activity in vitro against bacteria, fungi, and viruses, and may contribute to the "oxygen-independent" defense pathways of these cells. Lehrer, R.I., T. Ganz et al., "Oxygen-independent bactericidal systems. Mechanisms and disorders," Hematol Oncol Clin North Am, 2(1):159-69 (1988). Expression of defensin in a non-myeloid tissue source, the mouse small intestinal crypt cells, has also been reported. Ouellette, A.J., R.M. Greco et al., "Developmental regulation of cryptdin, a corticostatin/defensin precursor mRNA in mouse small intestinal crypt epithelium," J Cell Biol, 108(5):1687-95 (1989).

Cecropins, magainins, and defensins all share the properties of being cationic and membrane active, and evidence suggests that their antimicrobial activity is secondary to their ability to selectively disrupt membranes, possibly by channel formation. Bevins, C.L. and M.A. Zasloff, "Peptides from frog skin," Ann. Rev. Biochem., 59:395-414 (1990); Kagan, B.L., M.E. Selsted et al., "Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes," Proc Natl Acad Sci USA, 87(1):210-4 (1990); Lehrer, R.I., A. Barton et al., "Interaction of human defensins with Escherichia coli. Mechanism of bactericidial activity," J Clin Invest, 84(2):553-61 (1989); Zasloff, M.A., "Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active froma, and partial cDNA sequence of a precursor," Proc Natl. Acad Sci USA, 84:5449-53 (1987).

These newly emerging family of basic, cysteine-rich peptides with antimicrobial activity found throughout the animal kingdom include the defensins (Ganz, T., M.E. Selsted et al , "Defensins," Eur J Haematol, 44(1): 1-8 (1990b)), insect defensins (Lambert, J., E. Keppi et al., "Insect immunity:isolation from immune blood of the dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides" [published erratum appears in Proc Natl Acad Sci USA May;86(9):3321(1989)]. Proc Natl Acad Sci USA 86(1): 262-6 (1989)), bactenecins (Romeo D., B. Skerlavaj et al., "Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils," J Biol Chem, 263:9573-75 (1988)), sapecins (Matsuyama, K. and Natori, S. "Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Saroophaga peregrina,"J biol Chem, 263:17112-16 (1988)) and royalisin (Fujiwara, S., J. Imai et al., " A potent antimicrobial protein in royal jelly," J Biol Chem, 265: 11333-37 (1990)). Defensins are basic peptides of 30 to 34 amino acids with 3 disulfide bonds. The known characterized defensins from both myeloid and non-myeloid tissues all have highly conserved amino acid residues within the family, including 6 invariant cysteines. Aside from a pair of cysteine residues near the carboxy-terminus of the tracheal antimicrobial peptide of this invention, no consensus or other residues are shared between these peptides. Furthermore, the 5' region of all known defensin cDNAs are strikingly conserved even across species, and no similarity with this consensus region is found in the tracheal antibiotic peptide's cDNA. Comparison with the other cysteine containing antimicrobial peptides shows no similarity.

Formal searches of the NRBF protein data base using a modification (IBI) of fastP, Lipmann, D.J. and W.R. Pearson, "Rapid and sensitive protein similarity searches,.revreaction. Science, 227:1435-41 (1985) and the Intelligenetics Search found no protein sequences that disclose the tracheal antimicrobial peptides of the instant invention. A nucleotide-based search of the GenBank data base using the University of Wisconsin Genetics analysis software, Devereux, J., P. Haeberli et al., "A comprehensive set of sequence analysis programs for the VAX," Nucl. Acids Res., 12:387-95 (1984), was similarly unrevealing.

There is provided by this invention a novel tracheal antimicrobial peptide (TAP) and a novel precursor of TAP.

There is provided by this invention a novel cDNA coding for a TAP and a novel cDNA coding for a TAP precursor.

There is provided by this invention a novel protein produced from the cDNA coding for a TAP and a novel protein produced from the cDNA coding for a TAP precursor.

There is provided by this invention a novel substantially purified, isolated mammalian TAP precursor comprising a protein having at least in part or in whole substantially the same amino acid sequence as the protein defined in SEQ ID NO:3.

There is provided by the invention a substantially purified, isolated mammalian TAP comprising a protein having at least in part or in whole substantially the same amino acid sequence and at least the same antimicrobial activity as the protein defined in SEQ ID NO:1.

There are provided novel methods of use and diagnosis for TAP and cDNA coding for TAP.

There are provided novel recombinant host cells transformed with DNA coding for TAP or a portion thereof sufficient for the expression of TAP by said host cells.

There is provided a novel method of producing TAP which comprises culturing recombinant host cells wherein a recombinant DNA transformed in said host cell has a DNA sequence encoding TAP, operably linked to appropriate regulatory control sequences which are capable of effecting the expression of said coding sequences in said transformed cells.

There is provided a novel recombinant vector capable of expression in a suitable expression system which comprises a DNA sequence encoding TAP operably linked to control sequences compatible with said expression system.

Applicants have found that the ciliated respiratory mucosa of mammals contains peptide-based antimicrobial activity, to complement other defense systems of the airway. An abundant novel peptide found in extracts of the mammalian tracheal mucosa, is isolated herein on the basis of potent antimicrobial activity. This molecule is hereinafter referred to as tracheal antimicrobial peptide (TAP).

FIG. 1. Purification of the antimicrobial peptide from Bovine trachea:

A. P-30 gel filtration chromatogram; fractions containing antimicrobial activity are marked.

B. Antimicrobial plate assay of P-30 fractions. 2.mu.l of each fraction was spotted on a lawn of E. coli strain D31 and incubated overnight at 37.degree. C.

C. Ion exchange HPLC chromatogram of P-30 15 antimicrobial fractions. Antimicrobial fraction eluting at 26 minutes is marked with an arrow.

D. Reverse phase HPLC chromatogram of the antimicrobial fraction from ion exchange HPLC.

FIG. 2. Amino acid sequence of TAP and related nucleotide sequences:

A. Amino acid sequence (SEQ ID NO:1) of a TAP based on a combination of peptide amino acid sequence analysis, mass spectral analysis, and cDNA sequence analysis. The arrows indicate the results from Edman degradation analysis: sequence 1 is from direct amino-terminal analysis, sequence 2 is following cyanogen bromide cleavage and HPLC purification. Cysteine residue were determined following reduction and treatment with 4-vinylpyridine.

B Nucleotide sequences (SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:12) of degenerate oligonucleotides used for PCR amplification. A 1:1 mixture of oligonucleotides was used as an upstream primer. Abbreviations used: Y=C,T; R=A,G; D=G,A,T; B=C,G,T. Lower case letters denote 5' flanking sequences included in the oligonucleotide to aid subcloning.

C. Nucleotide sequence of oligonucleotides (SEQ ID NO:8 and SEQ ID NO:9) used in cDNA screening. Selection of sequence was based on partial peptide sequence and codon usage tables.

D. Partial nucleotide sequence (SEQ ID NO:2) of bovine TAP cDNA obtained from dideoxy-sequence analysis.

FIG. 3. Nucleotide sequence (SEQ ID NO:4) and predicted amino acid sequence (SEQ ID NO:3) of a TAP precursor. Amino acid numbering starts with the initiation codon. The mature peptide (SEQ ID NO:1) beginning at residue 27 is underlined. The polyadenylation signal is boxed.

FIG. 4. Northern blot analysis of TAP (SEQ ID NO:1) message. Lane 1, 10.mu.g total bovine trachea RNA; lane 2, 10.mu.g total bovine lung RNA.

The term "antimicrobial" as used herein refers to killing microorganisms or suppressing their multiplication and growth.

As used herein, "TAP" refers to a protein having at least in part or in whole substantially the same amino acid sequence and at least the same antimicrobial activity as the protein defined in SEQ ID NO:1. Said antimicrobial activity being defined as substantially preventing the growth of E. coli K12 strain D31 under the conditions described below (TableII) at a concentration of at least 100 .mu.g/ml TAP. For convenience, a protein having at least in part or in whole substantially the same amino acid sequence as the protein defined in SEQUENCE ID NO:3 is hereinafter referred to as a TAP precursor.

The TAP protein of the invention, depending on the pH of its environment, if suspended or in solution, or of its environment when crystallized or precipitated, if in solid form, may be in the form of pharmaceutically acceptable salts or may be in neutral form. The free amino groups of the protein are, of course, capable of forming acid addition salts with, for example, organic acids such as hydrochloric, phosphoric, or sulfuric acid; or with organic acids such as, for example, acetic, glycolic, succinic, or mandelic acid. The free carboxyl groups are capable of forming salts with bases, including inorganic bases such as sodium, potassium, or calcium hydroxides, and such organic bases as piperidine, glucosamine, trimethylamine, choline, and caffeine. In addition, the protein may be modified by combination with other biological materials such as lipids and saccharides, or by side chain modification such as acetylation of amino groups, phosphorylation of hydroxyl side chains, or oxidation of sulfhydryl groups.

Modifications of TAP are included within the scope of the definition, so long as the antimicrobial activity as described herein is retained. Finally, it is understood that minor modifications of TAP may result in proteins which have substantially equivalent or enhanced antimicrobial activity as compared to the sequence set forth in SEQ ID NO:I. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental such a through mutation in hosts which are TAP producers. All of these modifications are included as long as the antimicrobial activity, is retained.

Acid extracts of mammalian tracheal mucosa, e.g. bovine, have been found to have the peptide TAP in abundant quantities, a peptide with potent antimicrobial activity (FIG. 1). A mammalian TAP (SEQ ID NO:1) was isolated by a combination of size-exclusion (FIG. 1A), ion-exchange (FIG. 1C), and reverse-phase (FIG. 1D) chromatographic fractionations using antimicrobial activity against a strain of E. coli as a functional assay. Purity of the isolated peptide (SEQ ID NO:1) was >95% as determined by a combination of analytical reverse-phase and capillary-gel electrophoresis (data not shown). The yield of the isolated material was 2 .mu.g/g of wet tracheal mucosa.

The mammalian TAP, SEQ ID NO:1, was characterized by amino acid sequence (data not shown), and compositional analysis (Table I) Mass spectral analysis determined the molecular weight of the peptide (SEQ ID NO:1) to be 4085 Da.

A cDNA (SEQ ID NO:4) corresponding to the precursor peptide, was cloned (FIG. 3), and contains an open reading frame of 64 amino acids (SEQ ID NO:3). It is believed that the thirty-eight carboxy-terminal residues of this open reading frame correspond to the isolated peptide (SEQ ID NO:1) based on several observations. First, the 33 amino acids determined from the amino acid sequence data align perfectly with residues 26-59 of the deduced sequence (FIG. 2A (SEQ ID NO:1) v. 2D (SEQ ID NO:2)). Second, the amino acid composition agrees favorably with the deduced amino acid sequence (Table I). Finally, the observed molecular ion of the isolated protein (SEQ ID NO:1), 4085 Da is in complete agreement with the deduced sequence, assuming the six cysteine residues all participate in intramolecular disulfide bonds. The predicted pI is 13.0, and there are no aromatic residues, both consistent with the observed protein data.

When assayed in vitro against several different strains of microbes, including some which are respiratory pathogens, a mammalian TAP (SEQ ID NO:1) of bovine origin showed similar inhibitory activity to that of synthetic magainin 2-NH.sub.2 (SEQ ID NO:5) , a naturally occurring antibiotic peptide from frog skin. See TableII. While TAP (SEQ ID NO:1) was most active against E. coli and K. pneumonia, significant antimicrobial activity was also seen when applied to C. albicans, thus TAP's spectrum of activity is believed to span at least to both bacteria and fungi.

Having described the amino acid sequence of a TAP (SEQ ID NO:1), including the precursor peptide (SEQ ID NO:3), it is believed these polypeptides can be routinely synthesized in substantially pure form by standard techniques well known in the art, such as commercially available peptide synthesizers and the like.

Additionally, it is believed TAP can be efficiently prepared using any of numerous well known recombinant techniques such as those described in U.S. Pat. No. 4,677,063 which patent is incorporated by reference as if fully set forth herein. Briefly, most of the techniques which are used to transform cells, construct vectors, extract messenger RNA, prepare cDNA libraries, and the like are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, for convenience, the following paragraphs may serve as a guideline.

Procaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species by Bolivar, et al, Gene (1977) 2:95. pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides additional markers which can be either retained or destroyed in constructing the desired vector. Commonly used procaryotic control sequences include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., Nature (1977) 198:1056) and the tryptophan (trp) promoter system (Goeddel, et al. Nucleic Acids Res (1980) 8:4057) and the lambda derived P.sub.L promoter and N-gene ribosome binding site (Shimatake, et al., Nature (1981) 292:128).

In addition to bacteria, eucaryotic microbes, such as yeast, may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's yeast, are most used although a number of other strains are commonly available. While vectors employing the 2 micron origin of replication are illustrated, Broach, J.R., Meth Enz (1983) 101:307, other plasmid vectors suitable for yeast expression are known (see, for example, Stinchcomb, et al., Nature (1979) 282:39, Tschempe, et al., Gene (1980)10:157 and Clark, L., et al., nz (1983) 101:300). Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess, et al., J Adv Enzyme Req (1968) 7:149; Holland, et al. Biochemistry (1978) 17:4900). Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeman, et al., J Biol Chem (1980) 255:2073), and those for other glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization (Holland, ibid). It is also believed terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 (Holland, M.J., et al., J Biol Chem (1981) 256:1385) or the LEU2 gene obtained from YEp13 (Broach, J., et al., Gene (1978) 8:121), however any vector containing a yeast compatible promoter, origin of replication and other control sequences is suitable.

It is also, of course, possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms. See, for example, Tissue Cultures, Academic Press, Cruz and Patterson, editors (1973). Useful host cell lines include VERO, HeLa cells, and Chinese hamster ovary (CHO) cells. Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) Fiers, et al., Nature (1978) 273:113), or other viral promoters such as those derived from polyoma, Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses. General aspects of mammalian cell host system transformations have been described e.g. by Axel; U.S. Pat. No. 4,399,216. It now appears, also that "enhancer" regions are important in optimizing expression; these are, generally, sequences found upstream or downstream of the promoter region in non-coding DNA regions. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes. Plant cells are also now available as hosts, and control sequences compatible with plant cells such as the nopaline synthase promoter and polyadenylation signal sequences (Depicker, A., et al., J Mol Appl Gen (1982) 1:561) are available.

Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, S.N., Proc Natl Acad Sci (USA) (1972) 69:2110, or methods described in Molecular Cloning: A Laboratory Manual (1988) Cold Spring Harbor Press, could be used for procaryotes or other cells which contain substantial cell wall barriers. Infection with Agrobacterium tumefaciens (Shaw, C. H., et al., Gene (1983) 23:315) is believed useful for certain plant cells. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology (1978) 52:546 can be used. Transformations into yeast can be carried out according to the method of Van Solingen, P., et al., J Bact (1977) 130:946 and Hsiao, C.L., et al., Broc Natl Acad Sci (USA) (1979) 76:3829.

cDNA or genomic libraries can be screened using the colony hybridization procedure. Generally, each microtiter plate is replicated onto duplicate nitrocellulose filter papers (S&S type BA-85) and colonies are allowed to grow at 37.degree. C. for 14-16 hr on L agar containing 50 .mu.g/ml Amp. The colonies are lysed and DNA fixed to the filter by sequential treatment for 5 min with 500 mM NaOH, 1.5M NaCl, and are washed twice for 5 min each time with 5xstandard saline citrate (SSC). Filters are air dried and baked at 80.degree. C. for 2 hr. The duplicate filters are prehybridized at 42.degree. C. for 6-8 hr with 10 ml per filter of DNA hybridization buffer (5xSSC, pH 7.0 5.times.Denhardt's solution (polyvinylpyrrolidine, plus Ficoll and bovine serum albumin; 1.times.=0.02% of each), 50 mM sodium phosphate buffer at pH 7.0, 0.2% SDS, 20 .mu.g/ml Poly U, and 50 .mu. g/ml denatured salmon sperm DNA).

The samples can be hybridized with kinased probe under conditions which depend on the stringency desired. Typical moderately stringent conditions employ a temperature of 42.degree. C. for 24-36 hr with 1-5 ml/filter of DNA hybridization buffer containing probe. For higher stringencies high temperatures and shorter times are employed. Generally, the filters are washed four times for 30 min each time at 37.degree. C. with 2.times.SSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7, then are washed twice with 2.times.SSC and 0.2% SDS, air dried, and are autoradiographed at -70.degree. C. for 2 to 3 days.

Construction of suitable vectors containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.

Site specific DNA cleavage can be performed by treating the DNA with a suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g., New England Biolabs, Product Catalog. In general, about 1 .mu.g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 .mu.l of buffer solution. Incubation times of about one hour to two hours at about 37.degree. C. are workable, although variations can be tolerated. After each incubation, protein can be removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol followed by running over a Sephadex G-5 spin column. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations can be found in Methods in Enzymology (1980) 65:499-560.

Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20.degree. to 25.degree. C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl,, 6 mM DTT and 5-10 .mu.M dNTPs. The Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present. If desired, selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the sticky ends. After treatment with Klenow, the mixture is extracted with phenol/chloroform and ethanol precipitated followed by running over a Sephadex G-50 spin column. Treatment under appropriate conditions with Sl nuclease results in hydrolysis of any single-stranded portion.

Synthetic oligonucleotides can be prepared by the triester method of Metteucci, et al. (J Am Chem Soc (1981) 103:3185) or using commercially available automated oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 0.1 nmole substrate in the presence of 50 mM Tris, pH 7.6, 10 mM MgCl:, 5 mM dithiothreitol, 1-2 Mm ATP, 1.7 pmoles .gamma..sup.32 P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.

Ligations can be performed in 15-30 .mu.l volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl,, 10 mM DTT, 33 .mu.g/ml GSA, 10 mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0.degree. C. (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt end" ligation). Intermolecular "sticky end" ligations are usually performed at 33-100 .mu.g/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations (usually employing a 10- 30 fold molar excess of linkers) are performed at 1 .mu.M total ends concentration.

In vector construction employing "vector fragments", the vector fragment can be treated with bacterial alkaline phosphatase (BAP) in order to remove the 5' phosphate and prevent religation of the vector. BAP digestions can be conducted at pH 8 in approximately 150 mM Tris, in the presence of Na+ and Mg+.sup.2 using about 1 unit of BAP per .mu.g of vector at 60.degree. C. for about one hour. In order to recover the nucleic acid fragments, the preparation is extracted with phenol/chloroform and ethanol precipitated and desalted by application to a Sephadex G-50 spin column. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.

For portions of vectors derived from cDNA or genomic DNA which require sequence modifications, site specific primer directed mutagenesis can be used. This is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation. Briefly, the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage.

Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The resulting plaques can be hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.

Correct ligations for plasmid construction can be confirmed by first transforming a suitable host with the ligation mixture. Successful transformants are selected by ampicillin, tetracycline or other antibiotic resistance or using other markers depending on the mode of plasmid construction, as is understood in the art. Plasmids from the transformants can then be prepared according to the method of Clewell, D.B., et al. Proc Natl Acad Sci (USA) (1969) 62:1159, optionally following chloramphenicol amplification (Clewell, D.B., J Bacteriol (1972) 110:667). The isolated DNA Is analyzed by restriction and/or sequenced by the dideoxy method of Snager, F., et al. Proc Natl Acad Sci (USA) (1977) 74:5463 as further described by Messing, et al., F. Supp. Nucleic Acids Res (1981) 9.309, or by the method of Maxam, et al., Methods in Enzymology (1980) 65:499.

In a third alternative, TAP can be prepared directly from a mammalian trachea as described below.

Also provided by this invention are methods of treating a microbial infection, such as bacterial or fungal, comprising administering to a mammal in need of such treatment an antimicrobially effective amount of TAP. Such treatment can be systemic or topical for treatment of e.g. acne, burns, eye infections, mouthwash, deodorant or topical fungicide. TAP could also be used as a contact disinfectant.

For use as an antimicrobial agent, TAP can be formulated into pharmacological compositions containing an effective amount of TAP and a usual nontoxic carrier, such carriers being known to those skilled in the art. The composition can be given via a route of administration suited to the form of the composition. Such compositions are, for example, in the form of usual liquid preparations including solutions, suspensions, emulsions and the like which can be given orally, intravenously, subcutaneously or intramuscularly. The composition can be administered in an antimicrobially effective amount, generally a dose of about 0.1 to about 100 mg/kg/day, calculated as protein is expected to be useful.

Other utilities of the invention disclosed herein include, use of the cDNA sequence of TAP as a marker for various genetic studies in the mammal. This type of marker can be used to diagnose genetic diseases which may be linked to this marker, if not directly due to a defect in the TAP gene. In addition, genetic markers such as this can be used in Restriction Fragment Length Polymorphism (RFLP) studies for breeding purposes as are other genetic markers.

Additionally, since TAP is found in the mammalian airway and may represent part of the host defense against infections, it is believed that overexpression of TAP is induced by the presence of certain microbes. Therefore, the presence of higher quantities of TAP may indicate the presence of specific organisms or the beginning of an infectious state. Quantitation of TAP protein by immunoassay or other such methods well known in the art, or measurement of TAP messenger RNA by well known hybridization techniques may serve as a diagnostic tool for infections.

All reagents were standard reagent grade from Baker, Philipsburg, NJ or Fisher, Pittsburg, PA unless otherwise noted. All bacteriological media were from Difco, Detroit, MI. Restriction enzymes were purchased from Bethesda Research Laboratories (Gaithersburg, MD) and were used according to manufacturer's protocol. Oligonucleotide probes were end labelled to a specific activity of ca. 10.sup.7 DPM/pmol using gamma-[.sup.32 P]dCTP (3000Ci/mmol, DuPont, Wilmington, DE) and T4 polynucleotide kinase (Stratagene, LaJolla, CA). Double-stranded DNA probes were labelled to a specific activity of ca 10.sup.9 DPM/.mu.g using alpha-[.sup.32 P]dCTP (800Ci/mmol), DuPont, Wilmington, DE) and T7 DNA polymerase with random oligonucleotide primers (Stratagene). Purified plasmid DNA was sequenced using the dideoxy-termination method with T7 DNA polymerase (U.S. Biochemicals).

A segment of adult bovine trachea (just proximal to the carina and approximately 40 cm in length) was obtained fresh from a local meat processing plant. The tissue was immediately placed on wet ice and processed within 2-3 hours. Preliminary experiments indicated that immediate processing of tissue gave no significant further improvement in yield. The epithelium and adherent connective tissue, dissected on ice from the underlying connective tissue and cartilage, was placed immediately in liquid nitrogen. The frozen tissue was then stored at -70.degree. C. for periods up to several months before further processing.

The frozen tracheal epithelium was pulverized with a mortar and pestle under liquid nitrogen. The frozen tissue powder was placed in boiling 10% (v/v) acetic acid, and boiling was continued for 10 minutes. The solution was allowed to cool to room temperature, and centrifuged at 23,000.times.g. for 30 minutes at 10.degree. C. The resulting supernatant was divided into 30 ml. aliquots and each aliquot was applied to a C18 Sep-Pak cartridge (Millipore Corp., Bedford, MA). This, and all subsequent procedures were performed at room temperature. The cartridges were washed with 0.1% trifluoroacetic acid (TFA) in H.sub.2 O (Buffer C) and then eluted with 4 ml. of acetonitrile/0.1% TFA (60:40, v/v; Buffer D). The cartridge eluates were dried, resuspended in 1-2 ml 6M guanidinium-HCl/20mM Tris-HCl, pH 7.4. The solution was applied to a Biogel P-30 column (40 cm..times.2.5 cm. diameter, Bio Rad) which had been pre-equilibrated with 50mM ammonium formate, pH 4.1. The exclusion limit was 40 kDa. The column was developed with the same ammonium formate buffer, and each fraction (2 ml.) was lyophilized, resuspended in water (0.1 ml.) and assayed for antimicrobial activity as described. Zasloff, M.A., "Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active froma, and partial cDNA sequence of a precursor," Proc Natl Acad Sci USA, 84:544953 (1987). The active fractions were pooled, and applied to a sulfoethyl ion exchange HPLC column (Poly LC, Columbia, MD). A 45 minute linear elution-gradient from buffer A to buffer B was employed at a flow rate of 1 ml./min. Buffer A contained 25% acetonitrile/5 mM potassium phosphate, pH 5.3, and buffer B was identical to buffer A except that it also contained 1 M NaCl. Preliminary experiments had established that only a fraction eluting at 26 minutes contained significant antimicrobial activity. In subsequent isolations, this fraction was applied to a reverse-phase HPLC column and fractionated using a linear gradient buffer C to buffer D at 1 ml./min. The peak fraction eluting at 28.5 minutes was lyophilized and resuspended in H.sub.2 O at an approximate concentration of 0.5 mg/ml.

The isolated peptide was subjected to amino acid analysis using an amino acid analyzer with automated hydrolysis (Applied Biosystems model 420, ABS, Foster City, CA). Sequence analysis was determined by the Edman degradation method on a pulsed liquid phase sequencer (Applied Biosystems model 477A, ABS). Cysteine residues were identified by sulfhydryl reduction followed by reaction with 4-vinylpyridine, Selsted M.E., S.S. Harwig et al., "Primary structures of three human neutrophil defensins," J Clin Invest, 76(4) 1436-9 (1985), prior to sequence analysis. The C-terminal portion of the peptide was isolated by HpLC following cleavage with cyanogen bromide. Matsudaira, P. "Limited N-Terminal Sequence Analysis," Guide to Protein Purification. Deutscher ed. Academic Press (1990). Cysteine residues were identified by sulfhydry reduction followed by reaction with 4-vinylpyridine prior to sequence analyses. Wilde, C.G., J.E. Griffith, et al., "Purification and Characterization of human neutrophil peptide 4, a novel member of the defensin family," J Biol Chem, 264 19 : 11200-3 1989).

Molecular weight of the unreduced peptide was independently determined by fast-atom bombardment on a JEOL HX110 mass spectrometer at 1000 resolution (Structural Biochemistry Center, University of Maryland, Baltimore County) and on a VG analytical ZAB 2-SE high field mass spectrometer operating at Vacc=8kv (MScan, Inc., West Chester, PA).

The pCR product was obtained using the degenerate oligonucleotide 5'-GAGCTCDGTICCDATYTGYTTCAT, SEQ ID NO:7, as an antisense primer, a 1:1 mixture of 5'-GAATTCAAYCCHGTBAGiTGYGTT, SEQ ID NO:12, and 5'- GAATTCAAYCCHGTBTCYTGYGTT, SEQ ID NO:6, as sense primers and a pool of bovine tracheal cDNA (prior to size fractionation) as a template. The general protocol for polymerase chain reaction (PCR) amplification Saiki, R.K., D.H. Gelfand et al., "Primer-directed enzymatic amplification of DNA with a Thermostable DNA polymerase," Science, 239:487-91 (1988) using reagents from a Gene Amp kit (Cetus) was modified for using the degenerate primers: final concentration template cDNA was 0.2ng/ml and of primers was 1.mu.M. After initial denaturation at 94.degree. C., reactions were incubated for 30 cycles of one minute at 94.degree. C., one minute at 55.degree. C. and three minutes at 72.degree. C. Bands were purified by electroelution after electrophoresis in polyacrylamide gels.

The techniques used for cDNA library construction have been described and reagents were from Invitrogen, San Diego, CA unless noted otherwise. Total mRNA was isolated from bovine tracheal epithelium tissue using according to the protocol of Chirgwin et al., "Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease," Biochem., 18:5294-99 (1979). Poly (A+) mRNA was selected using oligo dT cellulose columns (3'.fwdarw.5',Inc.). Poly A enriched RNA from bovine tracheal epithelium was reversed transcribed using a commercially available (Invitrogen) oligo dT primer. Second-strand cDNA was synthesized using RNase H digestion of the RNA-DNA hybrids and E. coli DNA polymerase I. T4 DNA polymerase was used to polish the blunt-ended cDNA, and then hemiphosphorylated, Not-1/EcoR-1 adaptors were added by blunt-end ligation. A portion (0.3.mu.g) of this cDNA was size-fractionated by agarose gel electrophoresis, and a fraction (300-3000 bp) was recovered by electroelution. The cDNA was ligated to EcoR-1 digested lambda gt10 (Stratagene) and the recombinant phage was packaged using Gigapak-Gold packaging extract (Stratagene). Approximately 5.times.10.sup.6 independent phage were obtained and 106 phage were plated at a density of 3.times.10.sup.4 /150mm plate on a lawn of C600 hfl.sup.- E. coli. Duplicate lifts were made using Colony/Plaque Screen filters (DuPont). The filters were screened sequentially using three probes: 5'-AATCCTGT AAGCTGTGTTAGGAATAAAGGCATCTGTGTGCCGAT-3', SEQ ID NO:10, 5'-AATAAAGGCATCTGTGTGCCGATCAGGTGTCCTGGAAGCATGAAACAGATTGG-3', SEQ ID NO:11 and the PCR product, PCR-BT40.1 (sequence not determined, the product was derived as detailed under PCR herein). The standard conditions for screening were modified: 50.degree. C., 6.times. SSC for hybridization and wash with the oligonucleotide probes; and 37.degree. C., 5.times. SSC/20% formamide and 55.degree. C, 6.times. SSC for hybridization and wash, respectively for the double stranded probe.

Selected clones were plaque purified, and DNA was obtained from liquid lysates. The cDNA inserts were obtained by digestion with EcoRI, and subcloned into Bluescript plasmid (Stratagene).

RNA was fractionated by agarose gel electrophoresis in the presence of formaldehyde and blotted to nylon membranes (Nytran, Schleicher and Schuell) by the capillary technique. Radioactive labelled DNA probes were hybridized to the immobilized RNA in 20% Formamide/5.times.SSC/5.times. Denhardt's/0.1% SDS at 42.degree. C., and washed in 0.1.times.SSC/0.1% SDS at 65.degree. C.

Antimicrobial activity during purification was determined by plate assay described in Zasloff, "Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active froma, and partial cDNA sequence of a precursor," Proc Natl Acad Sci, USA, 84:5449-53 (1987). A concentrated aliquot of each fraction (2-5ul) was spotted onto a lawn of E. coli strain D31 (Steiner, H.D., Hultmark, A. et al., "Sequence and specificity of two antibacterial proteins involved in insect immunity," Nature, 292: 246-248 (1981)) on a petri dish containing 10 g/l Bacto Tryptone, 5g/l Yeast extract, 0.75% agarose (Sigma), 25mM Tris, pH7.4, 50 mM NaF, and incubated overnight at 37.degree. C.

Minimal inhibitory concentration (MIC) of the peptide was determined by incubating 2.5.times.10.sup.4 bacteria in 0.25.times. Tripticase Soy Broth (TSB) with increasing concentrations of the peptide in 96 well microtiter plates (Corning), overnight at 37.degree. C.

Isolation of the peptide

The bovine tracheal epithelium was extracted in acid and size-fractionated by gel filtration on Biogel P-30 (FIG. 1A). The elution profile of the tracheal extract showed two peaks of U.V. absorbance (monitored at 220nm). Antibacterial activity was assayed using E.coli strain D31 (Steiner, H.D. Hultmark, A., et al., supra (1981)) for fractions 13 (void volume) to 60 (included volume); fractions 32-36 showed significant activity, as evidenced by clear zones of killing (FIG. 1B). These fractions corresponded to the peptide region (i.e., <5,000 Da) when analyzed by SDS-polyacrylamide gel electrophoresis and silver stained for protein (data not shown).

The antimicrobial fractions were pooled and then fractionated on ion exchange HPLC (FIG. 1C). An isolated peak at 25.6 minutes contained the only detectable antimicrobial activity. This peak was collected and further purified by reverse phase HPLC (FIG. 1D). There was a single superimposed peak of protein (arrow) and antimicrobial activity (data not shown) eluting at 37.3% CH.sub.3 CN. The overall yield was approximately 2.mu.g/g of epithelium (wet weight). The purity of the peptide was 95% as assayed by analytical HPLC and acid-polyacrylamide gel electrophoresis (data not shown) which showed a single, basic band.

The purified peptide was subjected to protein sequence analysis using automated liquid phase sequencing and amino acid compositional analysis (TableI). The amino-terminal residues were analyzed directly, and the more carboxy-terminal residues were analyzed following cyanogen bromide cleavage (FIG. 2A). The sequence determined by protein sequence analysis predicted a molecular weight of 3443. The molecular weight of the purified peptide was determined by mass spectroscopy analysis to be 4085.5, indicating that the protein sequence was incomplete. It was decided to deduce the remainder of the peptide sequence by analysis of a cloned cDNA.

Degenerate-oligonucleotide primers (SEQ ID NO:6, SEQ ID NO:12 and SEQ ID NO:7) were designed corresponding to amino acids 1-6(BT-40-1 and -2) and 21-26 (BT40-3) respectively as shown in FIG. 2B. Sense Primers 1 (SEQ ID NO:6) and 2 (SEQ ID NO:12) had the EcoRI recognition site incorporated on the 5' end, and antisense primer 3 (SEQ ID NO:7) had the Sstl recognition site on its 5' end. These primers were used in a PCR using bovine tracheal cDNA as template DNA and were expected to amplify the nucleotide sequence coding for amino acids 1-26. The principal DNA product was 90 bp in length, as expected based on peptide primary structure and the selected oligonucleotide primers. This indicated that cDNA encoding the peptide was present in the library, and yielded a DNA template for probe synthesis.

The cDNA library from bovine tracheal epithelium (approximately 10.sup.6 independent lambda gt10 phage) was screened using three different probes in parallel: the PCR product (no sequence, product obtained as per PCR described herein), and two "best-guess" synthetic oligonucleotide probes (SEQ ID NO:10 and SEQ ID NO:11), designed based on the peptide SEQ ID NO:1 (FIG. 2A) using published codon frequency tables. Lathe, R. "Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations," J MoI Biol 183: 1-12, (1985). Only clones which hybridized with two of the three probes (of which there were 15) were considered positive, and 7 of these were taken for further analysis. Several positive clones were plaque purified and the inserts were subcloned into Bluescript plasmid (Stratagene). All inserts were of approximately the same size, and DNA sequence analysis was performed on one of them. The sequence for the cDNA clone pBT40-4.4, SEQ ID NO:4, is shown in FIG. 3.

The cDNA sequence of the precursor protein (FIG. 3, SEQ ID NO:4) contains an open reading frame of 64 amino acids in length from the first ATG codon (base 35). The deduced amino acid sequence of SEQ ID NO:1 encoded by nucleotides 113-225 of SEQ ID NO:2, beginning with an N-terminal asparagine residue is in perfect agreement with the amino acid data (FIG. 2D). The open reading frame, which extends 5 residues beyond the most carboxy-terminal residue of the sequence elucidated by peptide analysis, is followed by an in-frame termination codon. The amino acid sequence of the peptide from this clone has a predicted molecular weight of 4091; if all cysteine residues are involved in disulfide bonds, this would reduce the predicted molecular weight to 4085, in complete agreement with the mass spectroscopic data. The amino acid composition of the predicted peptide agrees favorably with the peptide data.

RNA isolated from whole lung and isolated tracheal mucosa was subjected to northern blot analysis using the cDNA insert (SEQ ID NO:4) as a probe (FIG. 4). Under stringent conditions the cDNA (SEQ ID NO:4) probe recognized an abundant message of approximately 400 bp in bovine trachea mRNA, along with a less abundant species of the same size in bovine lung RNA. Both lanes had identical amounts of RNA as evidenced by ethidium bromide staining of the gel (data not shown) and by hybridization to a bovine alpha-butulin probe (FIG. 4).

The purified bovine TAP, obtained as described herein, was tested on several strains of bacteria to determine its antimicrobial activity in vitro. The results shown in Table II indicate that TAP (SEQ ID NO:1) has antimicrobial activity for both gram positive and gram negative bacteria. In addition, the peptide has significant activity against the fungus Candida albicans. The observed activity of the peptide, SEQ ID NO:1, is on the same order of magnitude as synthetic Magainin-2-NH.sub.2 (SEQ ID NO:5), in the assay used here.