PROTEINE PROTEASE INHIBITORS FROM STREPTOMYCES

Abstract: Protease inhibitors, LEP-10 and LTI, produced by Streptomyces.

Description: TITLE Protein Protease Inhibitors from Streptomyces FIELD OF THE INVENTION This invention relates to microbial protease inhibitors and, specifically, to protein protease ' inhibitors produced by Streptomyces, which are structurally related to Streptomyces Subtilisin Inhibitor, and cloned genes and uses thereof in recombinant DNA processes. BACKGROUND OF THE INVENTION Protein protease inhibitors appear to play a role in regulation of protease functions in living organisms and cells. Such protease inhibitors are widely distributed in animals and plants. Exemplary of these are alpha-1-antiprotease, soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor and antithrombin. Microbially produced protein protease inhibitors include a family of dimeric proteins, each 10 to 12 kilodaltons (ld). This family includes SSI, alkaline protease inhibitor (API-2) from _ ; _ griseoincarnatus, plasminostrep in (PSN) from S^ antifibrinoly icus and a protease inhibitor from Streptoverticilium cin amoneum. The inhibitors of the SSI family share extensive sequence homology, e.g., about 70% between SSI and PSN, but appear to have different protease specificities. See, generally, "Protein Protease Inhibitors - The Case of Streptomyces Subtilisin Inhibitor (SSI)", edited by Hiromi et al., Elsevier, 1985, pages 1-14, 139-161 and 365-395. Kakinuma et al., U.S. Patent 4,014,860, disclose PSN and a producing strain thereof. Protein protease inhibitors have medical application such as in treatment of lung tissue degradation caused by deficiency in alpha-1-antiprotease. Protein protease inhibitors can also be utilized to prevent protein degradation caused by proteases such as are present * in serum. Wilson, EP-A-113,319, report use of Erythrina trypsin inhibitor to * inhibit conversion σ,f one chain tissue plasminogen activator to the two chain form which occurs in a presence of serum. Streptomyces are an attractive host for production of desired polypeptides by recombinant DNA techniques because they possess the necessary cellular "machinery" to export proteins and because a great deal of experience in culturing Streptomyces for antibiotic production has been acquired. A problem which has been encountered in production of heterologous proteins in Streptomyces is protein degradation by endogenous proteases. A second problem which has been encountered is in obtaining export signal sequences which can be fused to heterologous coding sequences to direct export ox heterologous gene products. Furthermore, it is desirable to obtain regulatory regions, e.g., promoters, ribosome binding sites and transcriptional enhancing/stabilizing sequences which can be used to express heterologous coding sequences in Streptomyces at high levels. Such promoters are typicallyassociated with production of abundant mRNA and/or gene products. Brawner et al. , EP-A-187,630 disclose a Streptomyces beta-galacotosidase gene expression unit and use of the promoter and of the export signal sequence thereof to express and export heterologous gene products. It is an object of the present invention to provide novel protein protease inhibitors from Streptomyces■ It is a further object to provide small exported proteins, which are exported and which are produced in abundant amounts, and DNA coding sequences, export signals and regulatory regions therefor. SUMMARY OF THE INVENTION The invention is of novel protein protease inhibitors selected from the group consisting of Lividans Exported Protein (10 kd) (LEP-10) and Lo gisporus Trypsin Inhibitor (LTI). In further aspects of this invention, the invention is a recombinant DNA molecule comprising (i) the LEP-10 or LTI gene expression unit, (ii) the LEP-10 or LTI coding sequence (iii) the LEP-10 or LTI regulatory region (iv) the LEP-10 or LTI export signal sequence or (v) a hybrid coding sequence fused to a heterologous coding sequence and a microorganism or cell transformed therewith. DETAILED DESCRIPTION OF THE INVENTION LEP-10 and LTI are closely related, novel protein protease inhibitors. They are of about the same size as, and share sequence homology with, the SSI family of protease inhibitors. LEP-10 was originally identified by Coomassie Brilliant Blue staining of SDS-PAGE protein gels as a low molecular weight (about 10,000 daltons) exported protein present in the medium of a culture of Streptomyces lividans strain 1326. Amino acid sequence data obtained on peptides derived from a tryptic digest of LEP-10 suggested homology with PSN and SSI. Using the amino acid sequence of one of the LEP-10 tryptic peptides, oligonucleotide probes were prepared and used to identify DNA fragments present in Sj_ lividans 1326 chromosomal library which contained a putative LEP-10 sequence. Plasmids containing such putative sequences were used to transform S_;_ albus, which does not naturally produce LEP-10 and transformants were shown to express LEP-10. LTI was similarly originally identified on protein gels from a culture of S_;_ longisporus. Preliminary amino acid sequencing thereof indicated homology with LEP-10. Initial screening of a S. lonqispor s library with the LEP-10 oligonucleotide probes resulted in obtainment of chromosomal DNA fragments which hybridized to the LEP-10 probes but which did not code for LTI. A polyclonal antibody was raised against LTI,. The anti-LTI antibody reacted with protein produced by the S_^ longisporus but not with LEP-10. Then, by probing with polynucleotide fragments containing the LEP-10 coding sequence, a 2.1 kilobase pair (kb) Bam HI fragment of S. longisporus chromosomal DNA was identified as putatively containing the LTI gene. S_j_ lividans 1326, which does not produce LTI, was transformed with a plasmid comprising the 2.1 kb sequence and shown to produce LTI by reaction with the anti-LTI antibody. This procedure, which forms a part of this invention, comprises transforming a Streptomyces host with DNA fragments from another microorganism or cell known to produce an exported protein. Transformant clones are then incubated on an agar plate in contact with an adsorbent substrate, e.g., a nitrocellulose- filter having 0.2 urn pore size, to allow exported proteins to adsorb to the substrate. The substrate is then lifted and dried and assayed for reactivity with antisera specific to the exported protein by standard i munossay techniques. Clones of transformants which reacted with the antisera are selected. The DNA fragment introduced into the transformants can then be sub-cloned. Thus, this invention includes a method of cloning and identifying DNA sequences which code for an exported gene product which comprises: 1) isolating the gene product from a culture of a producing microorganism or cell and raising specific ° antisera thereto, 2) cloning fragments of DNA from the producing microorganism or cell into a non-producing strain of Streptomyces, 3) contacting the Streptomyces transformants from 5 step 2 with an adsorbent substrate and incubating the transformants for a time sufficient to allow proteins exported by the transformants to adsorb to the substrate and then 4) assaying the substrate for reactivity with the 0 antisera from step 1. * Similarly, LEP-10 and LTI coding sequences, or sequences for similar proteases, can be identified and isolated from microbial or cell sources by hybridization probing of chromosomal DNA fragments with single-stranded 5 LEP-10 or LTI coding sequence fragments. Such probe fragments are preferably of at least about 30 nucleotides in length. Thus, this invention also includes a method of identifying DNA coding sequences for protease inhibitors which comprises hybridizing fragments of the LEP-10 or LTI 0 coding sequences to chromosomal DNA fragments of a microorganism or cell. The LEP-10 and LTI protease inhibitors share homology with each other and with SSI and PSN. LEP-10 and LTI are roughly 80% homologous with each other and roughly 5 70% homologous with SSI and PSN. Following are the amino acid sequences of mature LEP-10 and mature LTI as determined by amino acid sequencing of tryptic digests and/or DNA sequence analyses. Amino acid analyses were carried out in a Beckman 890M amino acid sequenator (Beckman Instruments, Fullerton, California). The sequences of SSI and PSN from published reports are shown where they differ from LEP-10 and LTI. 10 LEP-10 NH2-SER-LEU-TYR-ALA-PR0-SER-ALA-LEU-VAL-LEU-TΗR-VAL- LTI NH2-ALA-SER-LEU-TYR-ALA-PR0-SER-ALA-LEU-VAL-LEU-THR-VAL- PSN NH2-GLY- -MET- SSI ASP-ALA-PRO-SER-ALA- 20 LEP-10 GLY-HIS-GLY-GLU-SER-ALA-ALA-THR-ALA-ALA-PRO-LEU-ARG-ALA-VAL- LTI GLY-HIS-GLY-THR-SER-ALA-ALA-ALA-ALA-ALA-PRO-LEU-ARG-ALA-VAL- PSN -ASN- -THR-VAL-ASN- -GLU- SSI -LYS- -VAL- -THR-THR- -GLU- 30 40 LEP-10 THR-LEU-THR-CYS-ALA-PRO-THR-ALA-SER-GLY-THR-HIS-PRO-ALA-ALA- LTI THR-LEU-ASN-CYS-ALA-PRO-THR-ALA-SER-GLY-THR-HIS-PRO-ALA-ALA- PSN -ASN- SSI -GLY-PRO- 50 LEP-10 ALA-ALA-ALA-CYS-ALA-GLU-LEU-ARG-GLY-ALA-HIS-GLY-ASP-PRO-SER- LTI ALA-LEU-ALA-CYS-ALA-ASP-LEU-ARG-GLY-VAL-GLY-GLY-ASP-ILE-ASP- PSN LEU-GLN- " *" -GLY- -PHE-ASP- SSI GLY-SER- -ASP- -ALA-ALA-VAL-GLY- -LEU-ASN- 60 70 LEP-10 ALA-LEU-ALA-ALA-GLU-ASP-SER-VAL-MET-CYS-THR-ARG-GLU-TYR-ALA- LTI ALA-LEU-LYS-ALA-ARG-ASP-GLY-VAL-ILE-CYS-ASN-LYS-LEU-TYR-ASP- PSN -THR-VAL-ARG-GLY-ASP- -ALA- -LYS-GLN-PHE-ASP- SSI -THR-ARG-GLY-GLU-ASP- -PRO-MET-VAL- -ASP- 80 LEP-10 PRO-VAL-VAL-VAL-THR-VAL-ASP-G Y-VAL-TRP-GLN-GLY-ARG-ARG-LEU- LTI ' PRO-VAL-VAL-VAL-THR-VAL-ASP-GLY-VAL-TRP-GLN-GLY-LYS-ARG-VAL- PSN * -LYS-ARG-VAL- SSI -LYS-ARG-VAL- 90 100 LEP-10 SER-TYR-GLU-ARG-THR-PHE-ALA-ASN-GLU-CYS-VAL-LYS-ASN-ALA-GLY- LTI SER-TYR-GLU-ARG-THR-PHE-GLY-ASN-GLU-CYS-VAL-LYS-ASN-SER-TYR- PSN -THR- -SER-TYR- SSI -VAL- -SER- -GLU-MET- -HIS- LEP-10 SER-ALA-SER-VAL-PHE-THR-PHE-COOH LTI GLY-THR-SER-LEU-PHE-ALA-PHE-COOH PSN GLY-MET-THR- -PHE-COOH SSI GLY-SER- -ALA-PHE-COOH The above sequence is substantially accurate and in any event, is not limiting of the invention. It is understood that other LEP-10 or LTI sequences may be - obtained due, for example, to variations which do not significantly affect activity, alternative processing or analytical error. Both LEP-10 and LTI have been shown to inhibit trypsin in a standard trypsin inhibition assay. See, Travis, Meth. Enzym. , ]30:755-765 (1981). Thus, both LEP-10 and LTI can be used in medical and other applications, in which trypsin inhibition, or other protease inhibition is desired. In comparison to aprotinin, LTI is a weak inhibitor of trypsin. This result is not surprising given that protease inhibitors tend to have different protease specificities, even among the SSI family of inhibitors. Trypsin inhibitory activity was determined in a chromogenic assay using KABI S2288 as substrate (HO-ILE-PRO-ARG-p-nitroanilide. ) Scisson of the ARG-p-nitroanilide bond releases p-nitroaniline which absorbs at 410 nm; thus, the extent of cleavage is measured by the increase in absorbance at 410 nm. This assay was used in qualitative manner to determine the inhibitory activity of LTI and LEP-10. Briefly, serial s - two fold ' dilutions of either conditioned medium ^ (CM * ) or ammonium sulfate concentrates (AS) of conditioned medium were mixed with 25 ng of trypsin and the substrate was added. After 30 min at room temperature, the assay was stopped by addition of acetic acid. The assay was carried out in microtiter plates, which were then read in an ELISA reader. Similar samples of PSN were included as controls. The LTI samples had a strong brownish-black pigment which gave a high background in the assay; however, the purified protein shows inhibitory activity as well. The data shown are the absorbances measured following cleavage of the chromogenic substrate by trypsin. The growth medium itself shows no activity (data not shown) . 1 Inhibitor Reciprocal CM CM AS AS AS Dilution LTI PSN LTI PSN LEP-1 No. Inh. 0.898 0.772 O ' .763 0.751 n.d. 5 1 0.170 0.010 0.081 0.018 0.070 2 0.195 0.046 0.087 0.021 0.091 4 0.187 0.046 0.078 0.023 0.130 8 0.182 0.053 0.081 0.024 0.165 16 0.187 0.058 0.092 0.036 0.219 i π 32 0.209 0.273 0.176 0.076 • 0.293 LEP-10 and LTI both showed high activity compared to PSN. However, because the dilutions used were all too low to reach a 50% inhibition level the activities cannot be 15 closely compared accurately. ("n.d." means more detected.) The proteins can also be used to inhibit endogenous Streptomyces or other proteases and thereby to inhibit degradation of desirable proteinaceous products, including heterologous proteins produced by recombinant 20 DNA techniques. For this purpose, one or both of the inhibitors can be added to a Streptomyces or other, e.g., E. coli, Bacillus, yeast, insect or mammalian, cell culture. Alternatively, Streptomyces strains or other hosts can be genetically engineered to produce one or both 5 inhibitors along with a protein of interest, as described further below. The proteins can also be used to inhibit degradation of proteins in protease-containing solutions and as laboratory reagents such as in an assay for 0 protease inhibition. LTI, for example, has been demonstrated to inhibit conversion of one-chain tPA to two-chain tPA. 5 The gene expression unit for LEP-10, that is, the DNA sequence containing the LEP-10 coding sequence and regulatory regions required for transcription and translation, was localized on a 4 kb Pstl fragment of S_ ; _ lividans 1326 chromosomal DNA and, furthermore, within a 2.97 kb BamHI-Pstl fragment thereof. The LTI gene expression unit was found to be present on a 2.1 kb BamHI fragment of S , longisporus chromosomal DNA. The LEP-10 and LTI gene expression unit can be further isolated by additional restriction endonuclease or endonuclease/exonuclease digests and cloning and expression of fragments thereof or by further DNA sequencing. By similar techniques, the coding sequence alone can be isolated and cloned, such ' as in an expression vector and the regulatory region alone can be isolated and cloned, such as in a promoter probe vector. More specifically, the coding sequence can be fused in-frame to a promoter in a plasmid. Such plasmid is used to transform a Streptomyces or other, e.g., E^ coli,- B. subtilis, yeast," insect or mammalian host. Such recombinant host is then cultured and the medium or cell extracts are screened for presence of the inhibito . Screening can be, for example, by gel electrophoresis of ' proteins, wherein the inhibitor can be detected as a 10 kd protein, by a trypsin inhibition assay, by immunodetection using anti-LTI or anti-LEP-10 antibody, by hybridization with LTI or LEP-10 probes or fragments, and/or by amino acid composition analysis or sequencing of putative LTI or LEP-10 proteins. Exemplary of promoters known to function in Streptomyces are the Streptomyces beta-galactosidase promoter, the leftward promoter of lambda (PL), the tyrosinase promoter and promoters of genes conferring antibiotic resistanc e such as erythromycin resistance, neomycin resistance, and thiostrepton resistance. The regulatory region of LEP-10 or LTI can be inserted into a promoter probe vector, that is, one having a coding sequence for a readily detectable phenotypic marker such that following insertion of a functional promoter upstream of the marker sequence the sequence is expressed. Exemplary of markers useful in Streptomyces are: antibiotic resistance markers, e.g., thiostrepton, kanamycin and the Streptomyces β-galactosidase. The LEP-10 and LTI regulatory regions can be used to express LEP-10 or LTI in the native gene expression unit or to express heterologous polypeptides or proteins in a hybrid gene expression unit as transcriptional or translational fusions. For example, the promoter of either LEP-10 or LTI can be fused in frame upstream of a coding sequence for vaccine antigens, e.g., hepatitis B surface antigen and rabies glycoprotein; or for pharmacologically active proteins, e.g., interleukins, plasminogen activators, other protease inhibitors such as alpha-1-antiprotease, tumor necrosis factor, Factor VIII and influenza NS1. The export signal of LEP-10 or LTI can likewise be isolated and ligated to a coding sequence for *a polypeptide which is not normally excreted, in frame and downstream of a promoter. Functional derivatives of each domain within the gene expression units of the invention, i.e., promoter functions, export functions, and protease inhibition functions, can be prepared by use of restriction endonucleases, random mutagenesis such as by ultraviolet irradiation and site directed mutagenesis such as by insertion, addition or substitutions of synthetic oligonucleotides. Such derivatives can readily be checked for effect on function. See, e.g., Davis et al. , "Alv. Bact. Genetics", Cold Spring Harbor Laboratory (198C); Miller, "Experiments in Molecular Genetics", Unit III, Cold Spring Harbor Laboratory (1972); Botstein et al. , Science 229:1193 (1985); and Estell et al., Science 233:659 (1986). Functional derivatives of the coding sequences, and of variant proteins produced thereby, are included within the scope of this invention. Functional derivatives of the proteins can also be prepared by directly altering the proteins. This can be accomplished by chemical means including cleavage to remove amino acids and insertion or addition of amino acids. Such chemically prepared derivatives can be checked for activity such as by a trypsin inhibition assay. Functional chemically prepared derivatives are also included in the scope of the invention. It appears that LTI is expressed as prepro LTI, having a signal sequence which apparently is cleaved on secretion and a pro sequence which is cleaved extracellularly. In S_;_ longisporus, two molecular weight species are secreted. The putative pro LTI has 6 additional amino acids at the N-terminus, compared to mature LTI. In S. lividans, two molecular weight species r e also observed,- but N-terminal amino acid sequence analysis indicates that the processing sites in S. lividans may be different than in S. longisporus. Following is the DNA sequence for the LTI coding sequence and for some upstream and downstream untranslated regions. The putative signal and extracellular cleavage sites in S_;_ longisporus are indicated in the sequence by slashes (/) . The putative signal and extracellular cleavage sites in S. lividans are 3 amino acids and 1 amino acid, respectively, downstream (3'). -140 -130 -120 -110 -100 Sstll -80 * * * * * 1 * TTCAACAOSC AAGG * -TACIG AAACACATCG GGICGAGGTG TIT-TC GCG GOGCTACATG Ct-TtXEACrC -70 -60 -50 -40 -30 -20 -10 * * * * * * * GQ3CTQ3Cα-l GTCOGGCACC AAACCGGAAC GGGTCGGCAC AOOTOGAAT CCTGOGGAAG GATGCACACA 10 20 30 40 50 * * * * * A-G CGG AAC ACC GOG CGC TGG GCA GCC ACC CIC GCC CIC ACG GCC ACC GCC GTC TGC met arg asn thr ala arg trp ala ala hr leu ala leu thr ala thr ala val cys 60 70 80 100 110 * * * * * GGA COC CIC ACE GGA GCC GCG CTC GCC ACC CO- GCC GCT GCT CCC GCC TCG CTC TAC gly pro leu thr gly ala ala leu ala Thr Pro Ala Ala Ala Pro Ala Ser Leu Tyr 120 130 140 150 Nσtl 170 Goc ccc T03 GCC CIG GIG CTC ACC CTC GGC CAC GGC ACA AGC GO- GCC GCC GOG GCC Ala Pro Ser Ala Leu Val Leu Thr Val Gly His Gly Thr Ser Ala Ala Ala Ala Ala 180 190 200 210 220 * * * * * CCG CTG <XG GCC GTC ACC CIG AAC 1GC GCC COS ACG GCC TCC GGA ACC CAT CCC GCC Pro Leu Arg Ala Val Thr leu Asn cys Ala Pro Thr Ala Ser Gly Thr His Pro Ala 230 240 250 260 270 280 * * * * * * gcn gen CTC GCC TGC GCC GAC CIG CGC GGS CTC GGC GCT GAC A C GAC GCC CTG AAG Ala Ala Leu Ala Cys Ala Asp Leu Arg Gly Val Gly Gly Asp lie Asp Ala Leu Lys 290 300 310 320 ■ 330 340 * =--- ' - * * * * GCC CGA GAC GGC GTG ATC TGC AM .AAG CIG TAC GAC CCG GTC CTC GTC ACG CTC GAC Ala Arg Asp Gly Val lie Cys Asn Lys Leu Tyr Asp Pro Val Val Val Thr Val Asp 350 360 370 380 390 * * * * * GGA CTC TGG CAG GGC AAG CGC GTC TCC TAC GAA CGG ACC TTC GGC AAC GAG TGC CTG Gly Val Trp Gin Gly Lys Arg Val Ser Tyr Glu Arg Thr Phβ Gly Asn Glu Cys Val 400 410 420 430 440 450 * * * , * * * AAG AAC TOC TAC GGG ACC AGC CIC TTC GCC,TTC TGA GAGA CCOGGATOKG CWCTCCIO-T lys Asn Ser Tyr Gly Thr Ser Leu Fhe Ala Phe — " 460 470 480 490 500 510 520 * * * * * * * GCACAOCIGA AGAGGCACGC AG-TOGQGAG TGGSAGICCC 1-1-_UUU TAOOOCTCGA TTCGAOGCnA It is to be understood that the above sequence is substantially accurate and, in any event, is not limiting of the invention. For example, the illustrated sequence may be incorrect in one or a few base pairs and/or amino acids. Also, other sequences which also code for LTI may exist or may be fabricated, which other sequences may differ in one or a few base pairs and/or amino acids. Such other sequences are expected to be at least about 90% homologous with the illustrated DNA sequence. Also, cleavage of the pre and pro sequences may actually occur at different or additional sites. The relative amounts of pro LTI and mature LTI can be manipulated. S_;_ longisporus cultured in trypticase soy broth buffered with 100 mM MOPS (4-morpholinepropane-sulfonic acid) (pH 7.0) buffer (Sigma Chemical Co., St. Louis, Missouri) for 48 to 78 hours yields predominantly pro LTI. Following is the DNA sequence of the LEP-10 coding sequence and for same upstream and downstream sequences. The signal cleavage site is indicated. As in the case of the sequence for LTI, illustrated above, the following sequence is substantially accurate and is not limiting oil the invention. _39o -380 -370 -360 -350, -340 * * * * * * GCλ CGG GGA TGT ACG GGA GTC GGG CGG AGG CCG CGA CCG AGG CCA GGG CGC GTC CCG _330 -320 -310 -300 -290 -280 . * * * * * GCG TGC ACC GAC AGC AGG TTG TCC ATC CGC GCC GCC ACC AGC TCC CGG TCG CGG CCC -270 -260 -250 -240 -230 * * * * * AGG TAG CCG GGG GCT CCA CGG AGT GCG TCA TCA GGT CCC AGC CGG TGA ACC TCG CCG -220 -210 -200 -190 -180 -170 * * * * * * GOC AGC CCC GCC TCG TCC TCC AGG AAC GCT AAT CAA GAA ACG CCC AGT TCC GGA CTT -160 -150 -140 -130 -120 -110 * * * * * * GGA ACG TTC TAA TTC TGT GAC TTC ACG CCA CTG ATT CAA TAC GCA AGG TTA CCG AAC -100 -90 -80 -70 -60 * * * * * ACC GTG GGG TCG AGA TGA GTT TGC GTG CCG GGA CTC GGC AGA CTC GCC GCT CCG GCA -50 -40 -30 -20 -10 1 * * * * * * CCG ACC GGG TGC ACC GGC ACC ACC CTC GAA ACG AGC GGA AGG ATG CAC ACA ATG CGG Met Arg 10 20 30 40 SO 60 a ■ a ■ • ■ AAC ACC GCC CCC TCG GCA GCC ACT CTC GGC CTG ACG GCC ACC GCC GTC TGC GGG CC C Aan Thr Al* Arg Trp Al* Al* Thr Leu Gly Leu Thr Ala Thr Al* Val Cye Gly Pro 70 80 90 100 Signal no 120 . . . • Cleavage . . CTC GCC GGG GCC TCC CTC GCC TCC CCG GCC ACC GCC CCC GCGJ TCG CTC TAC GCC CCC Leu Ala Gly Al* Ser Leu Al* Ser Pro λl* Thr Al* Pro λlajSer Leu Tyr Ala Pro 130 140 150 160 170 • •> * . . TCG GCC CTG GTG CTG ACC GTG GGG CAC GGA GAG ACC GCT CCC ACC GCC GCA CCC C TG Sec Ala Leu Val Leu Thr Val c y HI* Gly Glu Ser Ala Ala Thr Ala Ala Pro Leu l a 190 200 210 220 2 30 • . • • . . CGC GCC GTC ACC CTG ACC TGC GCC CCG ACC GCC TCC GGC ACC CAC CCG GCG GCC GCC Arg Ala Val Thr Leu Thr Cys Ala Pro Thr Al* Ser Gly Thr HI* Pro Ala Ala Ala * 240 2S0 260 2/0 2SO 2.0 • * . • * * GCG GCC TGT GCC GAA CTG CGC GCC GCG CAC GGC GAC CCG AGT GCC CTG GCC GCC GAG Ala Ala Cγa Ala Glu Leu Arg Ala Ala His Gly Asp Pro -βr Ala Leu Ala Ala Glu 300 310 320 330 * 340 . a a - * , GAC TCG GTG ATG TGC ACC CGG GAG TAC GCC CCC GTG GTC GTC ACC GTC GAC GCC GTC Asp Ser al Met Cye Thr Arg Glu Tyr Al* Pro Val Val Val Thr Val Asp Gly Val 150 360 370 380 390 400 . . a • • • TGG CAG GGG CGC CGC CTC TCC TAC GAA CCC ACC TTC GCC AAC GAC TCC GTG AAG AAC Trp Gin Gly Arg Arg Leu Ser Tyr Glu Arg Thr Phβ Ala Asn Glu Cys Val Lys Asn 410 420 430 440 450 460 GCC GGC AGC CCS AGC GTC TTC ACG TTC TCA GGG ACC GGG ACC GCC GGA CTG CGC GTC Al* Gly Ser Ala Ser Val Phβ Thr Ph* 470 ■ 480 490 300 510 . . . . • ATC GGC TGC TCG CTA CTG GCG AGT GCG AGC GCC GCC GTA CGG GTC CCC CGG GCC CGC S20 530 540 550 560 570 . . • a • • ACC GGG GAC GCC GGA CGG AGT GCG GCC GTC CGC CGT TCT CCC CGT TCA GGG GCA CGC 580 5.0 600 610 620 630 ■ a a a a . TCG GCC GTT CGC GGC CGG GCC GTC CGC GCC CCT GGC GGA CCG GGC ACG GTG GCT GTG 640 650 . a TCG CAC CAC GCA TAG CCC CGA CTG f- '._ ~ z"-T The LEP-10 and LTI proteins, as noted above, were originally discovered as products of S_ lividans 1326 and S. longisporus. It is likely, however, that other strains and species produce the same or substantially the same proteins, i.e., substantially homologous (>90%, especially >95%) and substantially the same substrate (protease) specificity and activity. Also, the LEP-10 and LTI proteins can be synthesized by standard protein synthesis techniques or DNA coding sequences therefor can be synthesized by standard DNA synthesis techniques. Thus, the invention includes the LEP-10 and LTI proteins and gene expression units and functional domains thereof regardless of source. Oligonucleotide probes for detecting similar DNA sequences in other strains and species can be synthesized based on the LEP-10 and LTI sequences. However, as noted above, results of probing with such oligonucleotides may produce false positives. Therefore, probing initially or secondarily with larger DNA fragments, e.gl, greater than about 30 nucleotides, and preferably greater than about 50 nucleotides, is preferred. By such probing techniques, genes encoding the same inhibitors or other inhibitors of the SSI family can be identified. By recombinant DNA techniques, the coding sequences of the invention can be used to produce large quantities of LEP-10 and LTI. These techniques comprise, in sum, transformation of a host, bacterial or eukaryotic, with a gene expression unit comprising the LEP-10 or LTI coding sequence of the invention. For this purpose, the native gene expression unit on a plasmid or other vector or a hybrid gene expression unit comprising the inhibitor coding sequence and a heterologous regulatory region can be used. Thε inhibitors so produced are purified to a desirable extent by standard protein isolation techniques. The following Examples are illustrative, and not limiting of the invention. EXAMPLES Example 1. LEP-10 S. lividans strain 1326 (Agricultural Research Culture Collection, Peoria, Illinois, NRRL 15091) was cultured in SL-glycerol, SL-glucose and YEMES broths at 28°C for approximately 30 hours. After spinning down the cells by low speed centrifugation, the supernatants were concentrated by ammonium sulfate precipitation, redissolved and electrophoresed on SDS-PAGE (15%) gels to separate protein products. Upon staining with Coomassie Brilliant Blue R-250, a dense band corresponding to a protein having a molecular weight of about 10,000 daltons was identified as having been expressed in certain broths. SL medium comprises components SL-A and SL-B and a trace elements solution as follows: SL-A (NH 4 ) 2 SO 4 1. .0 g/1 L-asparagine 2. 0 g/1 K 2 HPO 4 9. 0 g/1 NaH 2 P0 4 1. 0 g/1 SL- -B yeast extract 2C ) g/i MgCl 2 e6H 2 0 5. 0 g/1 CaCl 2 eH 2 0 0. l g/i trace elements so 2C I ml/1 Trace elements sol. ZnCl 2 40 mg/1 FeCl 3 §2H 2 0 200 mg/1 CuCl 2 e2H 2 0 10 mg/1 MMnnCCll 2 ,ee44HH 29 00 10 mg/1 Na2B4.O_7elOH2o 10 mg/1 (NH 4 ) 6 Mθ ? 0 24 e4H 2 0 10 mg/1 SL-A is autoclaved and SL-B is filter sterilized prior to adding together in a ratio of 1:10 SL-B: SL-A (v/v) . To prepare SL-glucose and SL-glycerol, 1% (w/v) glucose and glycerol, respectively, are added. Samples of the protein were cut out and removed from gels. The protein was reduced and S-dansylamidoethylated with dansylazridine. The alkylated protein was digested with trypsin and the tryptic peptides were recovered by reverse phase HPLC. Individual peptides were sequenced in a Beckman sequenator to obtain the primary structure data reported above. Based on the amino acid sequence, a series of single stranded oligonucleotide probes were prepared. Probe #263 was a mixed 24-mer as follows: G 3'CGGAAGCGCTTGCTCACGCAGTTC5' C T T C These probes were used to probe a Charon phage library of S_ ; _ lividans 1326 chromosomal DNA prepared substantially as described by Maniatis et al., "Molecular Cloning-A Laboratory Manual," 1982, Cold Spring Harbor Laboratory. The recombinant phage,,Charon 25.5 (Ch25.5) , contains Streptomyces lividans 1326 chromosomal DNA which hybridizes to the mixed oligonucleotide probe #263. This probe is complementary to the LEP-10 mRNA. A Bglll-EcoRI fragment ("about 18 kb) of Ch 25.5 was cloned into the Bam HI-EcoRI site of plasmid pUC18 to yield plasmid pDl. From pDl was isolated a Pstl fragment (about 4 kb) which also hybridized with the oligonucleotide probe. This Pstl fragment was cloned into the Pstl site of pBR322 to yield plasmid pBR33. The 4 kb Pstl fragment contains a BamHI-Pstl fragment (about 2.97 kb) fragment which hybridizes with oligonucleotide probe. Both the 4 kb Pstl and the 2.97 kb Bam Hl-Pstl fragments were cloned into the shuttle plasmid vector pMB157, which is a low copy number E_ ; _ coli-Streptomyces shuttle vector comprising SCP2 stability and replication functions, pUC8 sequences and thiostrepton and ampicillin resistance. The recombinant plasmids pMB157-21 and pMB157-8 (which contain the 2.97 kb BamHI- Pstl fragment) and pMB157-22 (which contains the 4 kb Pstl fragment) were transformed into protoplasts of S_;_ albus which was previously shown not to produce LEP-10. Colonies of transformants were checked for the production of LEP-10 protein by immunoblotting. Nitrocellulose filters (0.2 um) were applied directly to plates containing colonies of the transformants. After the filters were removed from the plates they were processed with antibody to LEP-10 by the Western blot procedure. Transformants of S_;_ albus 0 containing the recombinant plasmids produced extracellular LEP-10 as determined by this immunoblot procedure. LEP-10 in culture supernatants of S^ albus (pMB157-22) and S. albus (pMB157-21) grown in SL-glycerol medium was also detected by Western blots of SDS-PAGE gels. Thus, all of 5 the information necessary to produce a mature extracellular LEP-10 protein in S_j_ albus is present on the 2.97 kb Bam Hl-Pstl fragment. Partial sequence of the LEP-10 gene has been identified on an Rsal fragment (about 180 bp) isolated °. from ' the recombinant phage Ch25.5. This Rsal fragment is present in both the aforementioned 4 kb Pstl and the 2.97 kb BamHI-Pstl fragments. The Rsal fragment contains a sequence encoding the carboxy-terminal one-third of the LEP-10 protein. 5 Example 2. LTI Using the oligo LEP-10 probes described in Example 1 above, regions of homology were identified in chromosomal DNA of _;_ longisporus (ATCC 23931) previously shown to produce a small exported protein of about 10 kd 0 having N-terminal amino acid sequence homology with LEP-10. DNA sequence data subsequently showed that such DNA fragments did not code for a LEP-10-1ike protein. An alternative approach, involving cloning the LTI gene in Streptomyces and identifying the clones by screening with anti-LTI antibody, was employed. Antibody against purified LTI was raised in rabbits and found to react with LTI but not with LEP-10. This antibody was also found to react with protein produced by colonies of S. longisporus adsorbed to 0.2 um nitrocellulose filters following 4 hours incubation at 28°C but not with protein produced by S_ ι . lividans. Detection was enhanced by detection of the LTI-antibody complex using biotinylated goat anti-rabbit IgG and streptavidin-biotin-biotinylated horseradish peroxidase complex (a Vectastain kit, Vectastain Laboratories, Burlingame, California). For cloning, a 2.1 kb BamHI fragment was identified in S. longisporus DNA using three different LEP-10 fragment probes including the Rsal fragment. 2-2.3 kb sized BamHI fragments were cloned into plasmid pIJ703 (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) (Bglll-cut) and transformed into S lividans. Transformants were screened with anti-LTI antibody to identify a positive clone. (A higher frequency was obtained through use of a low copy number vector in a comparable experiment.) This positive clone was in a group of six colonies ' and had to be picked and ' rescreened. Plasmid DNA from positive colonies was isolated and shown to contain an insert of about 2.1 kb. Culture supernatants (trypticase soy broth, 2_ C, 48 hrs) from these positives were spotted on nitrocellulose filters as described above and probed with anti-LTI antibody and found to be positive. Transformation of _;_ lividans and of S_ ; _ albus, both of which were previously shown not to produce LTI, with plasmid DNA resulted in production of LTI as shown by western analysis and trypsin inhibition of S_ ; _ albus supernatants. Southern blots were probed with a DNA fragment containing the Rsal fragment from LEP-10 and hybridization was observed to the insert. LTI is harvested from Streptomyces longisporus conditioned medium by ammonium sulfate precipitation (65% saturation) . The precipitate, which floats, is collected by centrifugation which causes the "precipitate" to form a dense mat floating on the surface of the liquid. This mat is recovered and resuspended in 10 mM ammonium acetate pH 6.0, and dialyzed against 50 to 100 volumes of the same buffer for 16 to 18 hours. The dialyzed sample is harvested and the large amount of dark brown fibrous material remaining insoluble is removed by centrifugation. The clarified supernatant is applied to a carboxymethyl cellulose column (CM-52, Whatman) equilibrated in the dialysis buffer. After unbound protein is washed through, the column is developed with a linear gradient from 10 to 250 mM ammonium acetate, pH 6.0. The LTI elutes at approximately 150 mM ammonium acetate. The pooled LTI peak is dialyzed against 10 mM ammonium acetate, pH 6, and stored at 4°C. Example 3. Heterologous Gene Expression and secretion using LTI regulatory regions pLTI520 is pUC 18 (Yanisch-Perring et al. , Gene 3_3:103 (1985)) containing the entire LTI coding sequence and about 410 upstream base pairs on a 920 base pair • , Sacl-Kpnl fragment of S_;_ longisporus chromosomal DNA. A filled-in Banl fragment of the interleukin-1 beta (1L-1B) (Myers et al., J. Biol. Chem. (in press)), which lacks a regulatory region, was ligated to the LTI sequence at the Notl site (between bases 158 and 159 in the above- illustrated DNA sequence for LTI); the Notl site was blunt-ended by treatment with mung bean nuclease prior to ligation. A X nl-BamHI fragment of pLTI520, containing the LTI - IL-1B fusion, was inserted into pSK02 (Brawner et al., Gene 4):191 (1986)). The X nl site is in the ampicillin resistance gene in pUC 18. The BamHI site is in the polylinker region upstream of lac Z in pUC 18. The pSK02 derivative was transformed in a galK deficient mutant strain of S^ lividans 1326, strain 12K (Brawner et al. , cited above). After sporulation, transformants were inoculated into trypticase soy broth (TSB) and incubated at 28°C for at least 72 hours. Both supernatants and cell extracts were analyzed by SDS-PAGS and western immunoblotting. These analyses showed expression and secretion of two proteins, both of which reacted with polyclonal anti-IL-lB. One of the two appeared to be mature IL-IB. The other had a slower mobility consistent with that expected for IL-IB fused to 17 amino acids of mature IL-IB. Example 4. Heterologous gene expression and secretion using LEP-10 regulatory regions A LEP-10-IL-1B fusion was constructed in pUC 18. The fusion contained S. lividans DNA from a Hinfl site (between bases -132 and -133 in the above-illustrated sequence for LEP-10) to a cut-back Bgll site (between bases 85 and 86) . The blunt-ended Bgll site was ligated to a filled-in Aval site in pUC18. Then, a DNA fragment containing the Banl IL-IB fragment with a BamHI linker was ligated to a BamHI site in pUC 18 to yield the construction illustrated as follows: » • * ' 1 2 3 ' 4 . [***CTC GCC T|CC GGGI GAT CCG| GCA CCT***| wherein region 1 is derived from LEP-10, region 2 is derived from pUC 18, region 3 is from the linker and region 4 is the IL-IB coding sequence. Maintenance- sequences from the Streptomyces vector, pIJ102 (Kieser et al., Mol. Gen. Genet. 185:223 (1982)), were then inserted. Following transformation of S_^ lividans 12K and culturing transformants in trypticase soy broth, an extracellular protein which corresponds approximately to mature IL-IB was observed by western immunoblotting. Although N-terminal sequence analysis of the excreted protein has not been carried out, it appears that cleavage may occur between the first residue (alanine) and the second residue (proline) of IL-IB to remove the LEP-10 signal sequence. 1 Examples 4 and 5 demonstrate utility of the LEP-10 and LTI regulatory regions both to express heterologous gene products in Streptomyces and to export heterologous gene products which are not otherwise 5 exported. The above description and examples fully describe the invention and preferred embodiments thereof. The invention, however, is not limited to precisely the embodiments described but also include all modifications 10 coming within the scope of the claims which follow. 15 ■ 20 25 30 35

Claims: CLAIMS : 1. A protein protease inhibitor selected from the group consisting of LEP-10 and LTI. 2. The protein protease inhibitor of Claim 1 which is LEP-10. 3. The protein protease inhibitor of Claim 1 which is LTI. 4. A recombinant DNA molecule comprising the LEP-10 or LTI gene expression unit. 5. A host microorganism or cell transformed with the DNA molecule of Claim 4. 6. A recombinant DNA molecule comprising the LEP-10 or LTI regulatory region. 7. The DNA molecule of Claim 6 wherein the regulatory region is operatively linked to a heterologous coding sequence. 8. A microorganism or cell transformed with the DNA molecule of Claim 6. 9. A recombinant DNA molecule comprising the " LEP-10 or LTI export signal sequence. 10. The DNA molecule of Claim 9 wherein the export signal sequence is operatively linked to a promoter and to a heterologous coding sequence. 11. A host microorganism or cell transformed with the DNA molecule of Claim 9. 12. A recombinant DNA molecule comprising the LEP-10 or LTI coding sequence. 13. The DNA molecule of Claim 12 wherein the coding sequence is operatively linked to a heterologous regulatory region. 14. A host microorganism or cell transformed with the DNA molecule of Claim 12. 15. A recombinant DNA molecule comprising a hybrid coding sequence having a portion of the LEP-10 or LTI coding sequence fused to a heterologous coding sequence. 16. The DNA molecule of Claim 15 wherein the coding sequence is operatively linked to a heterologous regulatory region. 17. A host microorganism or cell transformed with the DNA molecule of Claim 15. 18. A method of cloning and identifying DNA sequences which code for an exported gene product which comprises: 1) isolating the gene product from a culture of a producing microorganism or cell and raising specific antisera thereto, 2) cloning fragments of DNA from the producing microorganism or cell into a non-producing strain of Streptomyces, 3) contacting the Streptomyces transformants from step 2 with an adsorbent substrate and incubating the transformants for a time sufficient to allow proteins exported by the transformants to adsorb to the substrate and then ' 4) assaying the substrate for reactivity with the antisera from step 1. 19. A method of identifying DNA coding sequences for protease inhibitors which comprises hybridizing fragments of the LEP-10 or LTI coding sequences to chromosomal DNA fragments of a microorganism or cell. 20. A process for preparing a recombinant DNA molecule comprising the LEP-10 or LTI gene expression unit which process comprises hybridizing chromosomal DNA to a probe based on the LEP-10 or LTI gene expression unit to identify chromosomal DNA regions which comprise the LEP-10 or LTI gene expression unit and then isolating the identified chromosomal DNA regions by restriction with a restriction endonuclease. 21. A process for preparing a recombinant DNA molecule comprising the LEP-10 or LTI regulatory region which process comprises hybridizing the chromosomal DNA to a probe based on the LEP-10 or LTI regulatory region to identify chromosomal DNA regions which comprise the LEP-10 or LTI regulatory region and then isolating the identified chromosomal DNA regions by restriction with a restriction endonuclease. 22. A process for preparing a recombinant DNA molecule comprising the LEP-10 or LTI coding sequence which process comprises hybridizing the chromosomal DNA to a probe based on the LEP-10 or LTI coding sequence to identify chromosomal DNA regions which comprise the LEP-10 or LTI coding sequence and then isolating the identified chromosomal DNA regions by restriction with a restriction endonuclease. 23. A process for preparing a recombinant DNA molecule comprising the LEP-10 or LTI export signal sequence which process comprises hybridizing the chromosomal f DNA to a probe based on the LEP-10 or LTI export signal sequence region to identify chromosomal DNA regions which comprise the LEP-10 or LTI export signal sequence and then isolating the identified chromosomal DNA regions by restriction with a restriction endonuclease. 24. A process for preparing a recombinant DNA molecule comprising a hybrid coding sequence having a portion of the LEP-10 or LTI coding sequence fused to a heterologous coding sequence which process comprises restricting a recombinant DNA molecule, having all or a portion of the LEP-10 or LTI coding sequence, at a point downstream of a portion of the LEP-10 or LTI coding sequence and ligating to said portion a coding sequence for a heterologous polypeptide. 25. A process for producing a polypeptide in Streptomyces which comprises ligating a DNA fragment which codes for the polypeptide to the LTI or LEP-10 gene expression unit or to a portion thereof to prepare a recombinant DNA molecule, transforming Streptomyces organisms with the recombinant DNA molecule and then culturing the transformed Streptomyces such that the polypeptide is expressed.

Inventors: BERKA THOMAS RODNEY, FORNWALD JAMES ALLAN, GORNIAK JOSELINA GATMAITAN, ROSENBERG MARTIN, STRICKLER JAMES EDWARD, TAYLOR DEAN PERRON, BERKA, THOMAS, RODNEY, FORNWALD, JAMES, ALLAN, GORNIAK, JOSELINA, GATMAITAN, ROSENBERG, MARTIN, STRICKLER, JAMES, EDWARD, TAYLOR, DEAN, PERRON
Applicants: SMITHKLINE BECKMAN CORP, SMITHKLINE BECKMAN CORPORATION
Application year: 1987
Main classification: C07K13
IPC codes: C07K14, C12N15