ACTIVE SITE MODIFIED PROTEASE ALPHA-1-ANTITRYPSIN INHIBITORS AND THEIR PRODUCTION

Abstract: Proteinaceous compositions are provided which inhibit naturally occurring serine proteases. Particularly, an amino acid sequence analogous to human α₁-antitrypsin is modified at the active site while maintaining protease inhibition. The methionine at the active site is substituted with an oxidatively stable amino acid, while other amino acids may also be changed, added or deleted, particularly at the termini. The proteinaceous compositions can be produced in yeast, particularly the S. carlsbergensis/S. cerevisiae hybrid strain AB110 (containing plasmid pC1/1GAPATi9) deposited at the A.T.C.C. on May 9, 1984, and given Accession No. 20709.

Description: BACKGROUND OF THE INVENTION Field of the Invention Proteases or peptidases have a wide range of activities, functions and specificities in vivo . The need to produce various polypeptides for specific functions, while controlling their lifetime is an important function of peptidases. Peptidases also serve to process various proteins, whereby leader sequences may be removed, hormones or other active polypeptides excised from a larger polypeptide or the like. An alternative method for controlling the enzymatic activity at a particular site or in a medium in a host is the production of a specific enzyme inhibitor. This strategy is employed with human leukocyte elastase, a serine protease. Leukocyte elastase is involved in the phagocytosis of pathogens in the lungs. Failure to inhibit the enzyme, so that native tissue is attacked, can result in pulmonary emphysema or acute respiratory distress syndrome. The naturally occurring human α₁-antitrypsin has a methionine at the active site for inhibition of many serine proteases, particularly elastase. Oxidation of the methionine to the sulfoxide substantially inactivates the inhibitory activity towards most serine proteases, but not human chymotrypsin. The ability to inhibit an enzyme, either reversibly or irreversibly, has a wide variety of applications. For therapeutic purposes, the ability to protect a host from the degradative effects of elastase can provide protection from the diseases indicated above. In in vitro systems, there are also uses for inhibition. For example, in diagnostic assays, where one can modulate inhibition in relation to the presence or absence of a ligand, one can use the inhibition for a measurement of the presence or amount of the particular ligand. Furthermore, specific inhibitors can be used for titration of the enzyme, where only a non-specific substrate is available, for isolation and/or purification of an enzyme. Thus, novel enzyme inhibitors can have a wide variety of applications, both in vivo and in vitro . Description of the Prior Art Partial nucleotide sequences of α₁-antitrypsin have been reported by Kurachi et al ., Proc. Natl. Acad. Sci. USA (1981) 78 :6826-6830 and Leicht et al ., Nature (1982) 297 :655-659. See also Suggs et al ., Proc. Natl. Acad. Sci. USA (1981) 78 :6613-6617; and Ish-Horowicz and Burke, Nucleic Acids Research (1981) 9 :2989-2998. Carrell et al ., Nature (1982) 298 :329-334 provide the amino acid sequence of human α₁-antitrypsin, with discussion of various known antitrypsin mutants. Bollen et al ., DNA (1983) 2 :255-264 describe the cloning and expression in E . coli of a full cDNA coding for human α₁-antitrypsin. Courtney et al ., Proc. Natl. Acad. Sci. USA (1984) 81 :669-673 describe the expression of human cDNA for α₁-antitrypsin in E . coli . Kawasaki et al ., "The Production of Human α₁-antitrypsin in Yeast and Fission Yeast" in The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, 1983, describe the expression of human α₁-antitrypsin in S . cerevisiae and S . pombe employing the yeast triosephosphate isomerase promoter and terminator sequences. The protein is reported to be unglycosylated, but biologically active against trypsin and elastase. The existence of polymorphisms in α₁-antitrypsin is reported by Fagerhol and Cox, Adv. Hum. Genet. (1981) 11 :1-62. Beatty et al ., J. Biol. Chem. (1980) 255 :3931-3934, report that oxidation of α₁-antitrypsin reduces serine protease inhibitory activity. Nakajima et al ., ibid (1979) 254 :4027-4032 show that a valine derivative is a human leukocyte elastase substrate. Travis and Salvesen, Ann. Rev. Biochem. (1983) 52 :655-709, give a review of α₁-antitrypsin and its properties. EP-A-0 155 188 describes α₁-antitrypsin mutants produced in bacteria and yeast wherein the active methionine is replaced with alanine, valine, glycine, phenylalanine, arginine or lysine. McRae et al. , Biochemistry (1980) 19 : 3973-3978 relates to studies on the reactivity of various serine proteases with sequences related to the reactive side of α₁-antitrypsin, including sequences where the active methionine is replaced by valine, alanine and phenylalanine. SUMMARY OF THE INVENTION Novel polypeptide serine protease inhibitors are provided, demonstrating enhanced storage and oxidative stability with high levels of inhibition. Efficient production of the inhibitors is obtained in yeast, particularly hybrid yeast strains, where the polypeptide inhibitors may be isolated, purified and employed in a variety of ways. DESCRIPTION OF THE SPECIFIC EMBODIMENTS Novel DNA constructs are provided for expression of novel serine peptidase inhibitors which find use in in vivo and in vitro applications. The peptidase inhibitors share a substantial identity with human α₁-antitrypsin, while differing at the active site in having an oxidation-resistant amino acid, capable of simulating the inhibitory effect of methionine, and having from zero to five number percent of the other amino acids modified, deleted or added, usually not more than about ten amino acids, more usually not more than about five amino acids, from the naturally occurring wild-type α₁-antitrypsin, where each deletion, substitution or addition is counted as one. The compositions are found to have inhibitory activity to human leukocyte elastase comparable to the naturally occurring α₁-antitrypsin having methionine at the active site. The polypeptide products of the subject invention may be glycosylated or non-glycosylated, usually non-glycosylated and are available using hybrid constructs involving combining DNA sequences from diverse sources. Hybrid polypeptides may be prepared which will generally range from about 350 to 500 amino acids. These polypeptides will, except for the modification of the methionine residue, retain the α₁-antitrypsin sequence for an oligopeptide sequence of at least about 10 amino acids, usually at least about 40 amino acids, and more usually at least about 80 amino acids, which sequence includes the active site of α₁-antitrypsin and at least five amino acids flanking both sides of the active site. Where using the numbering of α₁-antitrypsin as indicated in the Experimental, this will involve amino acids 290 to 394, more usually 310 to 390 and particularly 340 to 380. The non-α₁-antitrypsin residues may be employed for immunogenic purposes, for modifying the characteristic of the inhibitory properties, or to provide a plurality of activities. Fragments may also find use, particularly fragments involving at least about 20 amino acids, more usually at least about 30 amino acids, and preferably at least about 50 amino acids, and not more than about 200 amino acids where the fragments include the active site and for the most part the naturally occurring amino acids on each side of the active site, generally at least about ten amino acids on each side, more usually at least about 15 amino acids on each side, where the amino acids do not differ by more than ten number percent, more usually by not more than about five number percent from the wild-type sequence provided in the Experimental section. It should be understood that to the extent that naturally occurring alleles have been already isolated or may be isolated in the future, those sites in α₁-antitrypsin where allelic variation is observed may also be varied in the subject polypeptides. Particularly, the residues of the sequence in the Experimental differing from those residues described by Carrell et al ., Nature (1982) 298 :329-334 and Bollen et al ., DNA (1983) 2 :255-264 may be modified with substantial retention of enzyme inhibition. The DNA and polypeptide sequences of particular interest are set forth in the Experimental section, where the modified active site is indicated. The substitution at the active site will be an oxidative-resistant L-amino acid of from three to six carbon atoms, preferably of from four to five carbon atoms, where the side chain may be hydrocarbon or hydroxy substituted hydrocarbon, preferably hydrocarbon, including alanine, valine, leucine, isoleucine, serine, and threonine, particularly valine. The DNA constructs which are employed will usually involve an extrachromosomal element having a replication system recognized by at least one unicellular microorganism, such as a bacterium, e.g., E . coli , B . subtilis , etc.; fungi, particularly yeast, e.g., S . cerevisiae , S . pombe , S . carlsbergensis , etc.; or other organism, e.g., Streptomyces , Neurospora , or the like. Conveniently, there may be two replication systems, so that a shuttle vector is provided, so that during the preparation of the vector, the construction may be conveniently cloned in E . coli for enhancing the amount of material available and purification of the construct. Alternatively, the gene may be introduced into the host under conditions which result in integration, particularly in conjunction with a gene which provides for amplification, e.g., dihydrofolate reductase, metallothionein, etc. The replication system may be obtained from either plasmids or viruses and may be derived from such plasmids as ColE1, e.g., pBR322, R-plasmid, e.g., pRK290, 2µm plasmid, pJDB219, YEp4, YEp24, etc. Many of the replication systems are generally available and have been extensively described in the literature. Usually the replication system will be available in combination with from zero to three, more usually from one to three genetic markers, structural genes which allow for selection of the host containing the replication system and marker, which combination will be referred to as a vector. The markers may allow for selection in one or more hosts or different markers may be used for the different hosts. The markers conveniently include biocide resistance, e.g., antibiotic resistance to streptomycin, penicillin, chloramphenicol, tetracycline, tunicamycin, kanamycin, etc.; heavy metals, e.g., mercury and copper; toxins, e.g., colicin; immunity; prototrophy in an auxotrophic host, e.g., uracil, leucine, histidine, tryptophan, etc. In addition, there will be transcriptional and translational initiation and termination regulatory sequences. These sequences may be available from a wide variety of genes, where the untranslated upstream and downstream domains or regions may be employed as a source for the necessary regulatory sequences. Of particular interest is the use of yeast as a host, where various transcriptional initiation regions may be employed, such as the transcriptional initiation regions of yeast glycolytic enzymes, e.g., glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, phosphoglucoisomerase, triosephosphate isomerase, phosphofructokinase, pyruvate kinase, phosphoglycerokinase, glucose-6-phosphate dehydrogenase, or the like; acid phosphatase; galactokinase; heat shock proteins; metallothionein; etc. The regions will generally range from about 200 to 1000bp, more usually about 200 to 600bp, and may extend into the coding region of the particular structural gene associated with the transcriptional initiation region. To that extent, the 5'-terminus of the peptidase inhibitor structural gene may include from zero to ten, more usually from zero to five, conveniently one to three codons of the structural gene associated with the regulatory region. Since the first codon will be the methionine initiation codon, the difference between the subject peptidase inhibitors and the wild-type peptidase inhibitors will involve the substitution of the first codon for methionine or presence of one or more codons, usually not more than about five, inserted between the methionine initiation codon and the second codon of the wild-type peptidase inhibitor α₁-antitrypsin. The transcriptional initiation region will normally include the sequences necessary for translational initiation as well. Thus, by employing the region involving transcriptional initiation, one also provides for translational initiation and expression. The transcriptional initiation region can be selected or modified, so as to be constitutive or regulated. Thus, one can choose a promoter which provides for constitutive transcription of the structural gene. Alternatively, one can select a promoter or modify a promoter to provide for induced or controlled expression of the structural gene. The promoter region of yeast glycolytic enzymes structural genes, as well as other structural genes, appears to be divided into two domains, one domain proximal to the structural gene which involves the binding of RNA polymerase and initiation of transcription, and a second domain distal to the structural gene which provides for enhanced efficiency of transcription and/or regulation. Therefore, by modifying the nucleotides beginning at about from about 300 to 500 nucleotides upstream from the initiation of the structural gene and extending at least to 200 nucleotides and usually 300 nucleotides or more in the upstream direction, one can modify the manner of expression. Thus, one can introduce at this site, sequences associated with regulation, such as the regulatory regions for such structural genes as GAL1, PHO5, or GAL10 or one can use temperature-sensitive regulatory regions, which regions may include the RNA polymerase binding domain. With these regions, one would employ a host which had the associated mechanism for regulation of the particular region. The termination region will be at least about 200 nucleotides and may be 300 or more and can be conveniently derived from the same structural gene as the promoter or a different structural gene. It is found that the termination region and initiation regions should be balanced, since strong promoters appear to require strong terminators for efficacy. Therefore, for the most part, terminators will be chosen from structural genes which have similar levels of expression in the host as the structural gene associated with the transcriptional initiation region. Where a cassette is available providing for transcriptional initiation and termination and one or more restriction sites intermediate the two regions, the subject structural genes may be inserted downstream from the transcriptional and translational initiation region, so as to be under the regulatory control of this domain, and followed by the termination region. Alternatively, one can join the various fragments to provide a construct comprising the transcriptional and translational initiation region, the structural gene having its initiation codon, and the transcriptional and translational termination region, which may then be introduced into an appropriate site of a vector having the appropriate replication system(s) and markers. In order to provide for the modified polypeptides of the subject invention, a variety of techniques may be employed for providing the varied codons necessary for producing the modified polypeptide. One technique is in vitro mutagenesis. In this technique, a single strand of the structural gene or fragment thereof may be cloned in a single-stranded virus, DNA or RNA. A primer is then hybridized to the single-stranded virus containing the structural gene of the α₁-antitrypsin, where the primer differs from the natural strand in the nucleotides to provide for the modified codon(s). Usually, the primer will have at least about ten nucleotides, more usually at least about 15 nucleotides and usually fewer than 50 nucleotides, more usually fewer than about 40 nucleotides, conveniently from about 25 to 35 nucleotides. Conveniently the virus M13 may be employed containing the α₁-antitrypsin structural gene or fragment thereof, which fragment includes the active site Particularly, where there are convenient available restriction sites bordering the active site, this fragment may be employed for mutagenesis, followed by joining to the other fragments to create the modified serine protease inhibitor. Mutagenized phage may then be detected and isolated employing the mutagenic primer as a probe and as appropriate, the fragment may be joined to the other fragments or where the entire gene was employed, the entire gene may be inserted into the vector. Conveniently, one may digest the M13 replicative form DNA so as to provide for double-stranded DNA. Instead of using in vitro mutagenesis, one can employ chemical DNA synthesis, where the mutagenized sequence is synthesized and may then be inserted between the α₁-antitrypsin 5' and 3' fragments to complete the gene. Again, one would choose or create convenient restriction sites for insertion, where desirably at least one of the sites should provide for a cohesive terminus. Other techniques may also prove to be useful, as appropriate. The final construct will include from one to two replication systems of different unicellular microorganisms, e.g., bacterial and yeast; from zero to three markers, preferably from one to two markers, at least one common marker or one marker for each host for each replication system, and a cassette comprising a transcriptional initiation regulatory domain, followed downstream in the direction of transcription by the structural gene encoding for the modified serine peptidase inhibitor, followed by an appropriate termination system. This construct may then be used for introduction into an appropriate host, e.g., yeast. A suitable yeast host may be obtained by crossing different strains of Saccharomyces having a desired genotype, wherein one of the strains contains an expression plasmid which includes a structural gene of interest. The expression plasmid has an efficient transcriptional initiation system for transcriptional regulation of the structural gene. The resulting crosses are selected for enhanced production of the polypeptide of interest (e.g., as α₁-antitrypsin mutant). In this manner, novel strains are produced that are specifically adapted to the efficient and enhanced expression of a desired foreign protein. Of particular interest as the host is a cross between two strains, employing the common fermenting strain S . carlsbergensis having the appropriate auxotrophy, which may be a laboratory or industrial strain, mated with an S . cerevisiae strain having the appropriate genotype, so as to provide for a strain which can be selected having the desired properties. The two strains will usually have at least one common genetic lesion, so that the progeny will also have the same lesion. Particularly, the resulting strain should be pep⁻, particularly pep4⁻, and auxotrophic in a convenient metabolite, which can be complemented with an extrachromosomal marker, for example a cross between two different Saccharomyces strains, the carlsbergensis leu⁻ strain 2150-2-3 (available from Lee Hartwell, University of Washington) and the cerevisiae leu⁻ strain AB103.1, where one of the cell lines, particularly the AB103.1 cell line bears the pep4⁻ mutation and carries the expression plasmid. A strain was selected, "cured" of the plasmid and designated AB110 (ATCC accession number 20709), which has the following genotype: MAT alpha, ura 3-52, leu 2-04 or both leu 2-3 and leu 2-112, pep 4-3, his 4-508 [cir°]. In addition to the other functional sequences, as noted above, one or more genetic markers may be employed for selection and maintenance of the yeast strain. These markers may provide for protection from a biocide, e.g., antibiotic, toxin, or heavy metal; provide prototrophy to an auxotrophic host, e.g., the amino acid his , leu , or trp or other metabolite, e.g., ura , etc., gene; immunity; etc. Preferably the host cell phenotype will be caused by a mutation or lesion resulting in a metabolic block, i.e., in auxotrophy, which can then be complemented by the wild-type, functional, plasmid DNA which was introduced. Various techniques are available for transforming the host with the DNA construct, where spheroplasts may be employed with polyethylene glycol precipitated DNA, conjugation, transfection, transduction, or the like. See, for example, Hinnen et al ., Proc. Natl. Acad. Sci. USA (1978) 75 :1929-1933. The modified yeast host is then grown in an appropriate nutrient medium to provide for expression of the desired polypeptide. Conventional rich nutrient media may be used or any other medium in which yeast can be satisfactorily grown, lacking the appropriate metabolite(s). For induced expression, the host will be grown to high density under conditions where expression of the polypeptide is not induced and when the desired cell density is achieved, the conditions will be changed to induce expression. For constitutive expression, the host will be grown in a rich medium to ensure efficient production of the desired polypeptide. After sufficient time for the polypeptide to be produced, the cells may be isolated, lysed by any convenient means, e.g., mechanical shearing or detergent, and the polypeptide of interest isolated and purified by conventional techniques, e.g., chromatography, such as affinity chromatography, electrophoresis, density gradient, crystallization, ultrafiltration, or the like. Where the polypeptide is secreted, the cells may be separated from the supernatant and the supernatant treated as described above. Of particular interest is the use of the glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase promoter regions having the RNA polymerase binding site and the transcription initiation site. In conjunction with these promoter regions or proximal domains are desirably used for regulated expression, the GAL4 regulator region or the PHO5 regulator region or a temperature sensitive regulator region. The terminator region may be derived from any convenient structural gene, particularly the glycolytic enzyme genes. While the exemplified construct does not involve the use of a leader sequence allowing for secretion, such a construct could be produced whereby the peptidase inhibitor would be secreted and isolated from the nutrient medium. See, for example, U.S. Patent Nos. 4,336,336; 4,338,397; and 4,411,994. See also U.S. patent application Serial No. 522,909, filed August 12, 1983. By employing the constructions of the subject invention, polypeptides may be produced in amounts usually at least about 1% of total soluble protein, more usually at least 2% of total soluble protein, and frequently at least 5% or higher. Where amounts of about 25% or higher of total soluble protein are attainable, the total soluble protein being the sum of total host and expression product soluble protein. The subject serine peptidase inhibitors are shown to have specific inhibitory activity toward elastase, as well as inhibitory activity toward other serine peptidases. The compositions have a minimum activity as compared to conventional elastase assays where inhibition by human α₁-antitrypsin is being measured of at least 25%, usually at least 35%, and desirably at least 50% of the α₁-antitrypsin (Met). The compositions are resistant to oxidation as compared to the naturally occurring active site containing methionine. For use in in vivo therapy, the subject compositions may be formulated in a variety of ways. Conveniently, the formulations may be liquid formulations involving deionized water, phosphate buffered saline, pH 7-7.5, or other physiologically acceptable medium. Usually, the composition will be administered by injection, either intraarterially or intravenously, or by inhalation employing an aerosol. Any dosage would depend on the manner of administration, the condition being treated, the frequency of administration, and the like, and would be comparable to the administration of enzyme inhibitory proteins, e.g., antibodies. The subject compositions can be used for affinity chromatography for isolating leukocyte elastase, by binding the subject compositions to an appropriate solid substrate, which may then be used in a column. Samples suspected of containing elastase may then be introduced into the column and eluted. By adding strong nucleophiles (e.g., hydroxylamine or hydrazine) to the eluting solution, the complex between the elastase and the subject serine peptidase inhibitors may be broken and the elastase eluted. Alternatively, various assays may be devised, whereby conjugating the subject inhibitors with a haptenic ligand of interest, the resulting conjugate may still retain inhibitory activity, which is lost when antibody to the hapten binds to the conjugate. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL All DNA manipulations were done according to standard procedures. See Molecular Cloning , T. Maniatis et al ., Cold Spring Harbor Lab., 1982. Enzymes used in cloning were obtained either from New England Biolabs or Bethesda Research Laboratories and employed according to the supplier's directions. Yeast were transformed and grown using a variety of media including selective medium (yeast nitrogen base supplemented with amino acids, etc.) as appropriate but without leucine, YEPD medium, containing 2% (w/v) glucose; and in the case of plating medium contained 2% (w/v) agar and for transformation 3% top agar. Cloning of alpha-1-antitrypsin . A cDNA library was made from 10µg of polyA⁺ RNA isolated from a part of a human liver. (Unless otherwise indicated α₁-antitrypsin is from a human source.) This library was prepared by oligo-dT priming of the first cDNA strand and self-priming of the second cDNA strand. The ds cDNA was size fractionated on a Sepharose CL4B column and those molecules greater than 300bp isolated. This fraction was treated with nuclease S1 and tailed with dCTP, using terminal transferase. The tailed cDNA was annealed to pBR322 which had been digested with Pst I and tailed with dGTP. Transformation of E . coli HB101 yielded 60,000 colonies, where greater than 90% of the clones were recombinant. Two synthetic oligonucleotide probes were used to isolate the alpha-1-antitrypsin (α₁-AT) cDNA, the first probe corresponding to amino acid residues 344-350 near the C-terminus of the protein was used to probe 5,000 colonies and the second probe, corresponding to amino acid residues -23 to -17 (+1 being the first nucleotide of the first codon of the mature α₁-AT) of the signal peptide, was used to probe 25,000 colonies. The probe sequences were taken from the partial nucleotide sequence described by Kurachi et al ., Proc. Natl. Acad. Sci. USA (1981) 78 :6826; Leicht et al ., Nature (1982) 297 :655). Approximately 3% of the colonies hybridized to the C-terminal probe and four hybridized to the N-terminal probe. The four N-terminal clones and 12 C-terminal clones were isolated and subjected to restriction analysis. From these, three overlapping clones which cover the entire cDNA were subjected to further study and were used to construct the full-length cDNA clone. The entire sequence of a composite full length cDNA derived from the three plasmids is as follows: LEGEND Nucleotide and predicted amino acid sequences of α₁-AT cDNA. The reactive center met-ser at positions 358-359 is boxed. Subscripts to amino acids in parentheses identify differences between the subject protein sequence and those derived from (a) protein sequencing (Carrell et al ., 1982), (b) the cDNA of Woo et al ., [see Carrell et al ., 1982]), and (c) the cDNA of Bollen et al ., 1983. The synthetic DNA molecules used in the construction of the Bam HI to Sal I fragment encoding the mature protein are shown as are the cDNA restriction sites used in this construction. The above sequence was determined using the dideoxy sequencing method of Sanger et al ., Proc. Natl. Acad. Sci. USA (1977) 74 :5463, in the M13 vectors of Messing et al ., Nucleic Acids Res. (1981) 9 :309. The differences at the nucleotide and amino acid level from the published cDNA sequences are shown. Construction of the full length clone for expression of yeast began with three fragments isolated from cDNA clones: 1) a 630bp Bam HI- Bst EII fragment; 2) a 450bp Bst EII- Ava I fragment; and 3) an 85bp Ava I- Hin fI fragment. A synthetic adapter was employed having the following sequence: Approximately two pmoles of fragments 1 and 2 were ligated together and after removal of the ligase, digested with Bam HI and Ava I. Fragment 3 and the synthetic adapter were ligated and digested with Ava I and Sal I and the two resulting fragment mixtures were ligated followed by digestion with Bam HI and Sal I. Fragments migrating upon electrophoresis in the region of about 1000-1400bp were isolated and cloned by substitution into Bam HI and Sal I digested and alkaline phosphatase treated pBR322. The resulting plasmid is referred to as pATi. Construction of pPGAP . A yeast expression vector was prepared called pPGAP having a polyrestriction site linker between the GAPDH terminator and a truncated GAPDH promoter region. Plasmids pGAP2 and pGAP1 were obtained as follows: A yeast gene library was prepared by inserting fragments obtained after partial digestion of total yeast DNA with restriction endonuclease Sau 3A in lambda-phage Charon 28 (Blattner et al ., Science (1977) 196 :161-169). The phage library was screened with DNA complementary to the yeast GAPDH mRNA and the yeast GAPDH gene GAP49 (Holland and Holland, J. Biol. Chem . (1979) 254 :5466-5474) from one of these clones was subcloned as either an about 3.3kb Bam HI fragment in the Bam HI site of pBR322 (pGAP-2) or as an about 2.1kb Hind III fragment in the Hind III site of pBR322 (pGAP-1). After digestion of the plasmid pGAP1 with Hin fI, a 500bp fragment was gel isolated, the fragment resected with Bal 31 to remove about 50bp, ligated with Hind III linkers, followed by digestion with Hind III and the resulting about 450bp fragment inserted into the Hind III site of pBR322 after treatment of the plasmid with Hind III followed by alkaline phosphatase. The resulting plasmid pGAP128 was digested with Hind III, the fragment made blunt-ended with the Klenow fragment of DNA polymerase I and nucleotide triphosphates and the resulting blunt-ended about 450bp fragment gel isolated and inserted into the Sma I site of plot5 after Sma I digestion and alkaline phosphatase treatment to provide the plasmid plot5pGAP128. plot5 was prepared by inserting the 40bp polylinker fragment of the following sequence into pBR322 as an Eco RI- Pvu II substitution followed by insertion of the trp - lac promoter (Russell and Bennett, Gene (1982) 20 :231-245) into the Pvu II site with transcription oriented toward the polylinker sequence. Plasmid plot5pGAP128 was then digested with Bam HI and Tag I to yield an approximately 390bp Bam HI- Taq I fragment having the -400 to -26bp of the GAPDH promoter. The Bam HI- Taq I fragment was ligated to a synthetic fragment having the following sequence: and containing -26 to -1bp of the GAPDH promoter and an Nco I site. The resulting Bam HI- Sal I fragment, which was digested with Bam HI and Sal I and used to replace the Bam HI- Sal I fragment of Bam HI- Sal I digested pBR322 treated with alkaline phosphatase. After ligation, the plasmid pGAPNRS was obtained which was digested with Bam HI and Sal I to provide a 400bp Bam HI- Sal I fragment which was gel isolated. This fragment was ligated to an about 1kb Sal I- Bam HI fragment containing the GAPDH terminator region and a short segment of 3' coding region and the resulting 1.4kb Bam HI- Bam HI fragment digested with Bam HI. The 900bp Sal I- Bam HI GAPDH terminator fragment was obtained by gel electrophoresis after Sal I and Bam HI digestion of pGAP2, prepared as described above. pBR322 was digested with Eco RI and Sal I, the termini blunt-ended and ligated to Bam HI linkers, followed by Bam HI digestion and the Bam HI- Bam HI 3.8kb fragment gel isolated, recircularized by self-ligation, cloned and designated pBRΔR1-Sal. The 1.4kb Bam HI- Bam HI fragment was inserted into the Bam HI digested, alkaline phosphatase treated pBRΔR1-Sal vector to provide the plasmid pPGAP of about 5.3kb with the orientation in the opposite direction of the amp r . Plasmid pPGAP was digested with Nco I, followed by blunt-ending, followed by Sal I digestion and treatment with alkaline phosphatase. The Nco I- Sal I fragment was substituted with an approximately 1250bp blunt-ended ( Bam HI)- Sal I fragment obtained from plasmid pATi, by Bam HI digestion, blunt ending, and Sal I digestion. This was inserted into the pPGAP vector to produce the plasmid pGAPATi, a 6.6kb plasmid, which was digested with Nco I and Bam HI and a 2.3kb Nco I -Bam HI fragment obtained having the α₁-AT gene and the GAPDH terminator and approximately 400bp Bam HI- Nco I fragment obtained having the GAPDH promoter. These fragments were ligated together and inserted into the Bam HI site of pC1/1. (Plasmid pC1/1 is a derivative of pJDB219 (Beggs, Nature (1978) 275 :104) in which the region corresponding to bacterial plasmid pMB9 in pJDB219 has been replaced by pBR322 in pC1/1.) Construction of pC1/1GAPATi8 and 9 The plasmids pC1/1GAPATi8 and pC1/1GAPATi9 (also referred to herein as pC1/1GAPATi(Met)) were obtained with the orientation of expression clockwise in the former and counterclockwise in the latter, with amp r being in the counterclockwise direction. One of these plasmids (Ti9) was transformed in S . cerevisiae AB103.1 (MATα, pep 4-3, leu 2-3, leu 2-112, ura 3-52, his 4-580 [cir°]) by standard methods (Hinnen et al ., Proc. Natl. Acad. Sci. USA (1978) 75 :1929-1933), selecting for leucine prototrophy and grown as described above. Yeast extracts were prepared by lysis with glass beads followed by centrifugation at 12,000 x g for 10min and the α₁-AT activity determined by inhibition of human leukocyte elastase. Assays contained in 1ml:0.1-0.2 human leukocyte elastase (HLE); 0.1mM MeO-Suc-Ala-Ala-Pro-Val- p -nitroanilide (Beatty et al ., J. Biol. Chem. (1980) 255 :3931); 50mM Tris, pH 8, 0.5M NaCl, and the indicated amounts of yeast extract or human α₁-AT. Assays were initiated by the addition of elastase, incubated at 28°C for 15min, terminated by the addition of 100µl of 8N acetic acid and the absorbance at 410nm determined. Typical results are shown in the following Table 1. The above data demonstrate that plasmids having the orientation of the expression cassette in the counterclockwise orientation, the promoter proximal to the long sequence of pBR322, make 10-20 times more α₁-AT than the same cassette in the other orientation. Yeast strain AB110 . Yeast strain S . carlsbergensis 2150-2-3 (available from Lee Hartwell, University of Washington) was crossed with a yeast S . saccharomyces strain AR103.1 transformant containing pC1/1GAPATi9. The diploids were sporulated and the tetrads dissected. Strains were maintained on leucine selective plates in order to ensure maintenance of the plasmid, since the parents are auxotrophs. A series of colonies were screened for their genotype with respect to a number of markers. The most vigorous strains were selected and cultures grown on leucine selective media. The best strain was designated AB110 (pC1/1GAPATi9) (ATCC accession number 20709), gave 6-7.5% of the total cell protein as alpha₁-AT as shown in the above Table 1. The strain AB110 has the following genotype: MATα, ura 3-52, leu 2-04 or both leu 2-3 and leu 2-112, pep 4-3, his 4-580 [cir°] and is obtained by curing the above strain AB110(pC1/1GAPATi9) of its resident plasmid by growth in the presence of leucine (absence of selective pressure) and then selection for leu⁻ colonies by replica plating. It is evident from the above results, that by employing a robust brewer's yeast S . carlsbergensis -baker's yeast S . cerevisiae hybrid strain as a host, auxotrophic as to a particular amino acid, for example, leucine, histidine, or tryptophan, and having a pep⁻ mutation particularly in the pep 4 gene, greatly enhanced yields of foreign proteins may be obtained. Desirably, after the original mating of the strains, the resulting pep4⁻ strains may be selected and crossed against the parent S . carlsbergensis strain with continuous selection for enhanced yields of the desired product. Mutagenesis of alpha-1-antitrypsin (ATi) . The Pvu II- Sal I fragment of the α₁-antitrypsin cDNA, described previously, coding for amino acids 238 to 394 was cloned by Sma I- Sal I substitution into M13 mp10 (Messing, Methods in Enzymology 101 part C, 20-78, (Eds. Wu, Grossman and Moldave) Academic Press (1983)). Single stranded viral DNA was isolated and used as a template for site directed mutagenesis as described in Zoller and Smith, Nucleic Acid Research (1982) 10 :6487-6500; Brake et al ., Proc. Natl. Acad. Sci. USA (1984) (described in U.S. patent application Serial No. 522,909, filed August 12, 1983), using a synthetic 30mer as a mutagenic primer. This primer of sequence 5' GACCTCGGGGGGGATAGACA C GGGTATGGC 3' was used to prime second strand synthesis on single-stranded M13 mp10 containing the wild-type α₁-antitrypsin fragment. The mismatch between wild-type fragment and primer is underlined in the primer sequence. Mutagenized phage were located using the same primer as a probe in hybridization experiments (Ish-Horowicz and Burke, supra ) and hybridized filters were washed for 60min at 65°C in 0.15M NaCl, 15mM sodium citrate. Single-stranded recombinant M13 mp10 was prepared from the positive phage plaques and the codon change in the α₁-antitrypsin fragment was confirmed by sequencing. Construction of pC1/1PHO5ATi(Val) and pC1/1PHO5ATi(Met) . Yeast expression plasmids containing the mutant α₁-antitrypsin gene (pC1/1PHO5ATi(Val)) or the wild-type α₁-antitrypsin gene (pC1/1PHO5ATi(Met)) under control of the PHO5 promoter and GAPDH terminator were constructed. Plasmid pC1/1PHO5ATi(Val) was constructed as follows. A 360bp Eco RV- Sal I fragment coding for amino acids 271 to 394 and containing the valine mutation was obtained by Eco RV- Sal I digestion of double-stranded M13 of the positively identified phage plaques containing the α₁-antitrypsin mutated fragment. This fragment was ligated to the 1350bp Bam HI- Eco RV fragment containing the PHO5 promoter and N-terminal residues of α₁-antitrypsin, prepared from pPHO5ATi, and to the ca. 1000bp Sal I- Bam HI GAPDH terminator fragment prepared from pPGAP. After ligation, the mixture was digested with Bam HI prior to ligation into Bam HI cut and alkaline phosphatase treated pC1/1. After transformation of E . coli HB101 one positive clone corresponding to pC1/1PHO5ATi(Val) was obtained after restriction analysis of plasmid DNA from 12 clones, with the orientation of ATi expression in the clockwise direction, with amp r being counterclockwise. Plasmid pC1/1PHO5ATi(Met) was constructed as follows. The Bam HI- Sal I 1800bp fragment containing the PHO5 promoter and the wild-type α₁-antitrypsin gene was prepared from pPHO5ATi. This fragment was ligated to the ca. 1000bp Sal I- Bam HI GAPDH terminator fragment prepared from pPGAP. After ligation, the mixture was digested with Bam HI prior to ligation into Bam HI cut and alkaline phosphatase treated pC1/1 to produce pC1/1PHO5ATi(Met), with the orientation of ATi expression in the clockwise direction, with amp r being counterclockwise. Plasmid pPHO5ATi was obtained as follows. The PHO5 gene was isolated from a yeast genomic library employing an oligonucleotide probe 5' GGCACTCACACGTGGGACTAG 3' derived from the published partial sequence (Meyhack et al ., The EMBO Journal (1982) 1 :675-680). A subfragment of this clone containing 550bp of the 5'-untranslated region and approximately 80bp of coding sequence was subcloned as a Bam HI- Sal I substitution in pBR322 to provide pPHO5. This fragment has the sequence 5' ATGTTTAAA 3' , encoding the first three amino acids, the second and third codons specifying an Aha III site. pPHO5 was digested with Bam HI and Aha III. The resulting 550bp fragment was isolated by gel electrophoresis and ligated to a ( Bam HI)- Sal I fragment isolated from pATi which contains the α₁-antitrypsin gene to produce a 1.7kb Bam HI- Sal I fragment. To obtain the ( Bam HI)- Sal I fragment, pATi was digested with Bam HI, the ends were blunted with Klenow, and the plasmid was subsequently digested with Sal I. The ( Bam HI)- Sal I fragment was purified by gel electrophoresis. The 1.7kb Bam HI- Sal I fragment containing the α₁-antitrypsin gene and PHO5 promoter was substituted into Bam HI- Sal I and alkaline phosphatase treated pBR322 to produce pPHO5ATi. Plasmid pPGAP was constructed as described under construction of pC1/1GAPATi(Val) and pC1/1GAPATi(Met). Plasmid pC1/1 is a derivative of pJDB219 (Beggs, Nature (1978) 275 :104) in which the region corresponding to bacterial plasmid pMB9 in pJDB219 has been replaced by pBR322 in pC1/1. The plasmid pC1/1PHO5ATi(Val) was transformed in S . cerevisiae AB103.1 (MATα, leu2 -3,112, ura3 -52, pep4 -3, his4 -580, [cir°]) (ATCC accession number 20711). The ABA103.1 line was used for other transformations as well (e.g., pC1/1PHO5ATi(Met)). Cells were grown in leucine⁻ selective medium in high or low phosphate. Construction of pC1/1GAPATi(Val) . Yeast expression plasmids containing the mutant α₁-antitrypsin gene (pC1/1GAPATi(Val)) under control of the GAPDH promoter and terminator were constructed. Plasmid pC1/1GAPATi(Val) was constructed as follows. A 600bp fragment corresponding to the 5' end of the α₁-antitrypsin gene was obtained by digesting pPGAPATi with Nco I and Bst EII and subsequent purification by gel electrophoresis. A second fragment of 470bp corresponding to the 3' end, including the active region of the mutagenized α₁-antitrypsin gene, was obtained by digesting pC1/1PHO5ATi(Val) with Bst EII and Sal I and further purification by gel electrophoresis. Both fragments were ligated together, and the resulting 1070bp Nco I- Sal I fragment containing the valine mutation was cloned into pPGAP, which had been previously digested with Nco I and Sal I and treated with alkaline phosphatase. The resulting plasmid, pPGAPATi(Val) was digested with Bam HI and the 2.6kb fragment containing the GAPDH expression cassette with the valine-α₁-antitrypsin gene, was purified by gel electrophoresis and inserted into Bam HI and alkaline phosphatase treated pC1/1. The plasmid pC1/1GAPATi(Val) was obtained with the orientation of ATi expression in the counterclockwise direction, the same as amp r . Plasmid pC1/1GAPATi(Val) was transformed in S . cerevisiae AB110 (Matα, ura3 -52, leu2 -04 or leu2 -3,112, pep4 -3, his4 -580, [cir°]) (ATCC accession number 20712). The AB110 line was used for other transformations as well (e.g., pC1/1GAPATi(Met)). The yeast cells were grown in leucine⁻ selective medium or in YEPD medium. Construction of pC1/1GAPATi(Ala/Pst) . A yeast expression plasmid containing a different mutant α₁-antitrypsin gene (pC1/1GAPATi(Ala/Pst)) under control of the GAPDH promoter and terminator was constructed. The plasmid was prepared essentially as described above for the other mutants, except that a Pst site was first incorporated 5' of Met₃₅₈ in the α₁-antitrypsin gene. The PstI site was added by M13 oligonucleotide site specific mutagenesis utilizing the following sequence as primer: This modified sequence was then cloned into pGAP. Thereafter, this plasmid was digested with PstI and SalI, yielding a fragment containing the 5' end of α₁-antitrypsin, the GAP promoter region and 280 bp of pC1/1. This fragment was isolated by gel electrophoresis. Separately, the pC1/1GAPATi(Met) plasmid was digested with AvaI and the following kinased 40/48 mer - containing an alanine codon (GCT) in place of the methionine codon at equivalent position 358 in the α₁-antitrypsin gene - was ligated to it. The linear fragment so-produced was subsequently digested with BamH1, yielding a PstI-BamH1 fragment containing the 3' end of α₁-antitrypsin as well as the GAP terminator. This fragment was purified by gel electrophoresis - and along with the other purified fragment was ligated into BamH1-SalI digested pC1/1, yielding pC1/1GAPATi(Ala/Pst). Anti-elastase activity and oxidation resistance of modified alpha-1-antitrypsin . Yeast extracts were prepared by lysis with glass beads and centrifuged to remove cell debris (Valenzuela et al ., Nature (1982) 298 :347-350). The soluble cell proteins were analyzed by SDS polyacrylamide gels (Laemmli, Nature (1970) 227 :680) and blotted onto nitrocellulose paper (Towbin et al ., Proc. Natl. Acad. Sci. USA (1979) 76 :4350-4355). α₁-antitrypsin-related proteins were visualized by washing the nitro-cellulose paper with rabbit anti-α₁-antitrypsin (Accurate Chemicals) followed by horseradish peroxidase/goat anti-rabbit antibody staining. Results of the gel analysis showed that a protein of 42,000 daltons is specifically induced in transformants containing the wild-type (Met) or mutant (Val) α₁-antitrypsin cDNA. This protein reacts with anti-α₁-antitrypsin. To determine the anti-elastase activities of the mutant (Val) or wild-type (Met) α₁-antitrypsin, appropriate yeast extracts were incubated with HLE and the synthetic peptide as described previously. Specifically for the assay, extracts were diluted in 50mM Tris pH8, 50mM NaCl. Aliquots were transferred to tubes containing 0.1mM Meo-Suc-Ala-Ala-Pro-Val- p -nitro-anilide (Vega), 50mM Tris pH8, 0.5M NaCl, 100µg BSA, in a volume of 1ml. Assays were initiated by the addition of 0.15µg HLE and were incubated for 15min at 28°C, terminated by the addition of 100µl of 8N acetic acid and the absorbance at 410nm determined (Beatty et al ., supra ). Protein concentrations were determined as described in Bradford, Anal. Biochem . (1976) 72 :248-253. From the amount of human leukocyte elastase activity observed, given the protein concentration of the yeast extracts and the amount of the enzyme added, the percentage of the yeast soluble protein which is α₁-antitrypsin can be calculated. It is assumed that there is 100 percent human leukocyte elastase activity, a 1:1 complex between human leukocyte elastase and α₁-antitrypsin whose molecular weights are 29kD and 42kD, respectively. In the experiment shown in Table 2 α₁-antitrypsin is 3.0 to 3.5 percent in the Met extract (assays 7 and 8) and 2.3 to 2.6 percent in the Val extract (assays 9 and 10). In Table 3, α₁-antitrypsin is 5.3% in the Met extract (assay 4) and 3.8 percent in the Val extract (assay 6). To determine if the valine-α₁-antitrypsin was oxidation resistant, extracts were treated with N-chlorosuccinimide (NCS) which converts susceptible methionines to their sulphoxides (Johnson and Travis, J. Biol. Chem. (1978) 253 :7142-7144). For this purpose, aliquots of the extracts were diluted in 50mM Tris pH8, 50mM NaCl, incubated at room temperature for 5min in 100µl of 50mM Tris pH8, with the indicated amount of NCS, and assayed for anti-HLE activity. Table 2 and Table 3 show results obtained with wild-type (Met) or mutant (Val) α₁-antitrypsin using pC1/1PHO5ATi vectors and pC1/1GAPATi vectors, respectively. Similar assays utilizing extracts from yeast transformed with the pC1/1GAPATi(Ala/Pst) plasmid gave comparable results, indicating the retention of activity of the mutant (Ala) α₁-antitrypsin. Effects of NCS Oxidation of α₁-antitrypsin Activity in Yeast Extracts Assayed by Inhibition of Elastase Activity Using pC1/1PHO5ATi(Val) or pC1/1PHO5ATi(Met) Assay No. Plasmid Vol. of Yeast Extract µg α₁AT NCS (10mM) Elastase Activity (%) 1 - - - - 100 2 - - 0.1 - 85 3 - - 0.2 - 67 4 - - 0.5 - 22 5 - - 1.0 - 0 6 C1/1 1.5µl - - 100 7 Met 0.5µl - - 54 8 Met 1.5µl - - 8 9 Val 0.5µl - - 52 10 Val 1.5µl - - 15 11 - - 1.0 - 5 12 - - 1.0 10µl 91 13 C1/1 3.8µl 1.0 - 12 14 C1/1 3.8µl 1.0 10µl 84 15 Met 1.5µl - - 8 16 Met 1.5µl - 10µl 93 17 Val 1.5µl - - 20 18 Val 1.5µl - 10µl 18 Effects of NCS Oxidation of α₁-antitrypsin Activity in Yeast Extracts Assayed by Inhibition of Elastase Activity Using pC1/1GAPATi(Val) or pC1/1GAPATi(Met) Assay No. Plasmid Vol. of Yeast Extract µg α₁AT NCS (10mM) Elastase Activity (%) 1 - - - - 100 2 - - - - 99 3 - - 1.0 - 10 4 Met 0.25µl - - 7 5 Met 0.5µl - - 6 6 Val 0.5µl - - 16 7 Val 1.0µl - - 13 8 - - 0.5 - 44 9 - - 0.5 + 96 10 Met 1.0µl - - 10 11 Met 1.0µl - + 53 12 Val 1.0µl - - 15 13 Val 1.0µl - + 15 14 Val 1.0µl 0.5 - 9 15 Val 1.0µl 0.5 + 15 It is evident from the above results, that the subject modified polypeptides are effective inhibitors of human leukocyte elastase and strongly complex with the elastase. The subject proteins can therefore find a wide variety of applications. Due to the substantially enhanced resistance to oxidation, as evidenced by the reaction with the N-chloro succinimide with retention of activity, the subject compounds can find use in commercial applications, such as affinity chromatography. Furthermore, by virtue of their enhanced stability in vivo , the compounds may find use in therapies for lung disorders. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims: Claims for the following Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE An S. carlsbergensis/S. cerevisiae hybrid strain obtained by crossing auxotrophic leu⁻ S. carlsbergensis yeast cells with non-complementing leu⁻ pep4⁻ S. cerevisiae transformed yeast cells and selecting for an auxotrophic leu⁻ S. carlsbergensis/S. cerevisiae hybrid strain providing for enhanced expression of at least a portion of the alpha₁-antitrypsin gene. A hybrid strain according to claim 1, which strain is AB110, A.T.C.C. Accession No. 20709 or 20712. A S. carlsbergensis/S. cerevisiae hybrid strain AB110, A.T.C.C. Accession No. 20709. The hybrid S. carlsbergensis/S. cerevisiae strain of claim 1 which is transformed with a vector encoding an analog of human alpha₁-antitrypsin wherein the amino acid corresponding to the methionine at position 358 of wild-type alpha₁-antitrypsin is substituted with an aliphatic amino acid, said analog exhibiting human leukocyte elastase inhibiting activity and resistant to oxidation by N-chlorosuccinimide. The hybrid S. carlsbergensis/S. cerevisiae strain of claim 4 wherein said aliphatic amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, serine and threonine. The hybrid S. carlsbergensis/S. cerevisiae strain of claim 5 wherein said aliphatic amino acid is valine or alanine. Claims for the following Contracting State : AT A process for producing an S. carlsbergensis/S. cerevisiae hybrid yeast strain comprising: crossing auxotrophic leu⁻ S. carlsbergensis yeast cells with non-complementing leu⁻ pep4⁻ S. cerevisiae transformed yeast cells; and selecting for auxotrophic leu⁻ S. carlsbergensis/S. cerevisiae hybrid cells. The process according to claim 1 wherein the hybrid strain is AB110, A.T.C.C. Accession No. 20709 or 20712. The process according to claims 1 or 2 wherein the S. carlsbergensis/S. cerevisiae hybrid strain is AB110, A.T.C.C. Accession No. 20709. The process according to claims 1, 2 or 3 wherein the hybrid strain is transformed with a vector encoding an analog of human alpha₁-antitrypsin wherein the amino acid corresponding to the methionine at position 358 of wild-type alpha₁-antitrypsin is substituted with an aliphatic amino acid, said analog exhibiting human leukocyte elastase inhibiting activity and resistant to oxidation by N-chlorosuccinimide. The process according to claim 4 wherein said aliphatic amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, serine and threonine. The process according to claim 5 wherein said aliphatic amino acid is valine or alanine.

Inventors: BARR, PHILIP J., BRAKE, ANTHONY J., HALLEWELL, ROBERT A., ROSENBERG, STEVEN
Applicants: CHIRON CORPORATION
Application year: 1985
Main classification: C07K15
IPC codes: C12N15, C07K14