IL92734A - Methods for producing polypeptides having protease inhibition activity of human mammalian alpha-1-antitrypsin and polypeptides produced thereby - Google Patents

Description

METHODS FOR PRODUCING POLYPEPTIDES HAVING PROTEASE INHIBITION ACTIVITY OF HUMAN MAMMALIAN ALPHA-1-ANTITRYPSIN AND POLYPEPTIDES PRODUCED THEREBY The present invention relates to a method of producing a polypeptide having the protease inhibition activity of human mammalian alpha-1-ant trypsin.

The present specification is divided from Israel Specification 69465, filed August 10, 1983.

In Israel Specification 69465 there is described and claimed a DtMA expression vector capable of replication in Saccharomyces cerevisiae and containing a Saccharomyces cerevisiae promoter which regulates the transcription of a glycolytic protein, which protein is triose phosphate isomerase or pyruvate kinase, said Saccharomyces cerevisiae promoter being followed downstream by a gene, under the regulation of said promoter, expressing a protein other than the glycolytic protein normally regulated by such promoter.

In order that the subject matter of the present invention may be better understood, the text from original specification 69465 has been retained herein, it being understood, however, that the subject matter claimed in said specification is specifically disclaimed and not included in the subject matter of the present invention.

The ability to obtain expression of foreign, i.e., exogenous, DNA in unicellular microorganisms provided the opportunity to conveniently prepare long polypeptide chains of interest. Almost immediately, varied polypeptides, such as the small hormone somatostatin and more sophisticated polypeptides, such as insulin, interferons, thymosin and a variety of vaccines having capsid proteins, were prepared and reported in the literature. For the most part, the initial work was performed in the bacterium E. coli which had been the subject of intensive study because scientists were familiar with many aspects of its genetic structure and properties. Initial attention was therefore directed to producing foreign proteins in E. coli. Once the ability to employ E. coli as a host was established, the limitations and disadvantages of employing E. coli encouraged the use of other hosts.

One host which appeared to be particularly attractive because it lacked many of the shortcomings of E. coli was yeast. However, yeast is a eukaryote and, therefore, has a more sophisticated genetic system. Furthermore, less is known about the yeast genome than is known about E. coli. In order to use yeast as a host for the production of proteins foreign to yeast, a number of discoveries are required, and new materials must be made available.

Initially, a replication system was required which provided stability in yeast, either as an extrachromo- somal element or by integration into the yeast chromosome. In addition, the regulatory functions concerned with transcription and expression had to be developed in order to allow for expression of the desired protein. There was also the uncertainty whether foreign DNA sequences would be transcribed and translated and, if expressed, whether the resulting polypeptides would survive in the yeast cell. Also remaining to be determined was the effect of the foreign proteins on the viability of the yeast cell, such as the effect of recombinant DNA (RDNA) on mitosis, sporulation and vegetative growth. - 2a - 92734/ 3 There have, therefore, been substantial efforts to develop novel RDNA systems in yeast, which will allow for regulated expression of a protein of interest, as well as highly efficient production of such proteins.

Hitzeman, et al., J. Biol. Chem. , 255:12073-12080 (1980) describe a plasmid having a yeast 3-phosphoglycerate kinase (PGK) gene and accompanying regulatory signals capable of expression in yeast. Other references of interest include Clifton, et al., Genetics, 88:1-11 (1978); Clark and Carbon, Cell, 9:91-99 (1976); Thomson, Gene, 1:347-356 (1977); Holland and Holland, J. Biol. Chem. , 254:5466-5474 (1979); Holland and Holland, ibid., 254:9830-9845 (1979); Nasmyth and Reed, Proc. Nat. Acad. Sci., 77:2119-2123 (1980); Broach, et al., Gene, 8:121-133 (1979); and Williamson, et al., Nature, 283:214-216 (1980).

According to the present invention there is now provided a method of producing a polypeptide having the protease inhibition activity of human mammalian alpha-1-antitrypsin, comprising the step of growing a yeast culture transformed by a DNA expression vector, said vector containing a promoter which regulates the transcription of a glycolytic protein, which protein is triose phosphate isomerase or pyruvate kinase, said promoter being followed downstream by a gene under the regulation of said promoter comprising a segment coding for human mammalian alpha-1-antitrypsin.

The present invention also provides substantially unglycosylated, active, human mammalian alpha-l-antitrypsin which is neither taught nor suggested by the prior art. Briefly, before the present invention, very few heterologous proteins had been successfully expressed in yeast hosts. Briefly, naturally-occurring mammalian alpha-l-antitrypsin is produced in mammalian cells as a precursor protein comprising a leader peptide which directs the protein molecule into the secretory pathway of the - 2b - 92734/ 2 mammalian cell which produces it. This leader peptide is then cleaved from the mature alpha-l-antitrypsin protein during processing in the secretory pathway. In addition, the naturally-occurring protein produced by mammalian cells is glycosylated. However, as described herein, an alpha-l-antitrypsin is produced which is substantially unglycosylated and lacks the leader peptide. This alpha-l-antitrypsin is not secreted from the yeast cell, but remains in the cytoplasm of the transformed yeast.

Prior to the present invention, the biological role of glycosylation upon proteins was not well understood. More specifically, in order for some proteins to be active, glycosylation is required,, while for others it is not. In addition, lack of glycosylation could alter the conformation of a protein, or expose otherwise protected regions to degradative enzymes within the yeast cell. Either of these events could result in the production of a protein without activity. Therefore, until one actually made the unglycosylated protein, one could not determine whether it would be active.

Various references have described expression of foreign proteins from eukaryotic cells. For example, Carbon, et al., "DNA Capable of Replication and Stable Mitotic Maintenance in a Host Eukaryote and an Eurkaryotic Cell Containing the DNA," EP 48,081, published March, 1982 (see also, CA 96:211842w) described hybrid DNAs (minichromosomes) that can be utilized for the stable transformation of eukaryotic cells. These minichromosomes comprise 3 components: (1) a centromere; (2) a DNA replication site; and . (3) one or more genes to be expressed.

Other authors have described the use of specific promoters such as the yeast alcohol dehydrogenase I promoters for expressing foreign proteins. In particular, Valenzuela, et al., "Synthesis and Assembly of Hepatitis B Virus Surface Antigen Particles in Yeast," Nature 298 ( 5872) : 347-50 (1982) (see also, - 2c - 92734/ 1 CA 98:3343y), describes the expression of a surface antigen of hepatitis B from the yeast Saccharomyces cerevisiae by utilizing an expression vector that employs the use of a 5' flanking region of yeast alcohol dehydrogenase I as a promoter to transcribe surface antigen coding sequences.

In like manner, Hitzeman, et al., "Expression of a Human Gene for Interferon in Yeast," Nature 293 ( 5835) :717-22 (1981) (see also CA 96:116850) describes the linkage of a DNA sequence coding for mature human leukocyte interferon D (Le-IF-D) with DNA fragments of the 5' -flanking sequences of the Saccaromyces cerevisiae alcohol dehydrogenase I gene on a plasmid. Yeast cells transformed with these plasmids synthesized biologically active LE-IF-D.

In contrast, the present invention provides methods of producing a polypeptide having the protease inhibition activity of human mammalian alpha-l-antitrypsin, comprising the step of growing a yeast culture transformed by a DNA expression vector, the vector containing a promoter which regulates the transcription of a glycolytic protein, which protein is triose phosphate isomerase or pyruvate kinase, the promoter being followed downstream by a gene under the regulation of the promoter, comprising a segment coding for human mammalian alpha-l-antitrypsin. The above-cited references do not suggest or disclose such methods.

In the accompanying drawings: Figs. 1A and IB are cDNA sequences of two forms of genes coding human alpha-l-antitrypsin. - 3 - Fig. 1 Illustrates the restriction maps of plasmids CTEA32 and CAT1.

Fig. 3 is a diagram of the electrophoresis chromatogram showing purified alpha-1-antitrypsin produced according to the present Invention.

Fig. 4 illustrates the restriction map of plasmid Cl/1.

Fig. 5 illustrates the DNA sequence of the multiple restriction site of pUC13.

Fig. 6 Illustrates the restriction map of plasmid pUC 1 containing the DNA sequence from Fig. 1.

Fig. 7 is a restriction map of plasmid HAT4.

Novel yeast promoters are provided which control the transcription of genes 1n the glycolytic pathway and which find use in the regulated production of proteins foreign to the yeast. Promoters of particular interest include the promoters for triose phosphate isomerase and pyruvate kinase. The protease inhibitor, mammalian alpha-l-antitrypsin, is expressed using the promoter for triose phosphate Isomerase.

Methods and compositions are provided for regulated efficient expression of alien or foreign DNA in a yeast host. (Alien or foreign DNA 1s DNA not naturally occurring in the wild type particularly from a different species and which does not normally exchange genetic information with the host). Novel promoters are employed which are Involved in the glycolytic pathway and provide for high levels of protein production, so that a 92734/2 - 4 -substantial proportion of the total protein produced by the yeast cells can be dedicated to the protein of interest. In addition, regulatory mechanisms associated with regulation of production of the glycolytic enzymes are achieved, so that production of the desired products may be modulated. Furthermore, viable cells can be maintained to enhance the efficiency and amount of expression.

The promoters of interest are particularly those promoters Involved with expression of trlose phosphate Isomerase, pyruvate kinase, phosphoglucose isomerase, phosphoglycerate mutase, hexokinase 1, hexokinase 2, glucoklnase, phosphofructo kinase, and aldolase, which are controlled by the glycolytic regulation gene GCR1.

The promoters may be obtained by employing a gene bank having large fragments of yeast DNA. By Introducing the fragments into appropriate vectors, particularly shuttle vectors having replicons for prokaryotes and yeast, one can readily amplify and clone the yeast DNA in a bacterium and then Introduce the yeast DNA Into mutant yeast cells for complementation. In this manner, yeast fragments can be Identified which complement auxotrophic lesions or mutations in a yeast host.

Of particular Interest, 1s where the host 1s auxotrophic in both the glycolytic pathway step of Interest and a separate biochemical pathway, which is complemented by a -5-marker in the vector. Once having established a DNA segment having the desired gene, one may redone by various techniques to shorten the DNA segment and provide for a segment which is primarily the gene of interest in conjunction with its regulatory signals for transcription and expression.

In order to retain the promoter, it is essential that the initiator methionine be determined and this codon be used for developing the strategy for introducing the alien DNA downstream from the promoter. Various techniques can be employed for providing a site for introduction of the alien DNA so as to be under the regulatory control of the promoter in the glycolytic pathway.

Where a restriction site is conveniently adjacent to the initiator methionine codon, the glycolytic gene may be cleaved at that site and the DNA chewed back with Bal31 for varying periods of time, so as to chew into or past the initiator methionine codon or retain the initiator methionine codon.

Where there is no convenient restriction site, other splicing techniques such as primer repair may be employed. Also, by employing in vitro mutagenesis, one can introduce a restriction site adjacent the initiator methionine, which encodes for the initial amino acids of the desired protein. In each instance, a linearized DNA segment is obtained having the intact promoter for the glycolytic product and normally includes other DNA sequences, such as an intact replicon, one or more markers, and the like.

Exemplary of the above procedure is the development of a vector having the promoter for the TPI1 gene. An exemplary vector CV13 having the replicons or replica- tion systems from pBR322 and 2j.-plasmid of yeast, as well as the LEU2 gene was employed for insertion of a yeast fragment which was shown to have the PI1 gene. This was achieved by employing double selection with a mutant yeast which was leu", tpi~. The TPI1 gene was found to have a unique Kpnl site. The vector was cleaved at the Kpnl site and then treated with the double stranded exonuclease Bal31 for varying times to chew back the DNA to about the f-met codon. Linkers were then inserted providing desired restriction sites. Alien DNA could then be inserted providing a sequence having a f-met codon in the appropriate position for initiation. Alternatively, the foreign DNA can be expressed using the f-met codon of the TPIl gene.

Similar procedures can be performed with the other subject glycolytic genes in order to provide the promoters associated with those genes. The PYK sequence has a convenient Xbal site for restriction, where the few additional bases may be removed, if required, using Bal31 for a short period of time to chew to or through the methionine codon. Of particular interest is the use of the GCR promoter to control the expression of the other genes involved in the glycolytic pathway. By employing the GCR gene, in conjunction with other glycolytic promoters regulating expression of alien DNA, one can turn on and off the other promoters, so as to regulate the expression of the alien DNA. Thus, one can allow vegetative growth to proceed until a desired cell density is achieved, before permitting production of the desired polypeptide.

By employing appropriate auxotrophs, one can further regulate the expression of the polypeptides of interest in choosing the appropriate nutrient medium. Where the chosen promoter is repressed by the particular nutrient because of a metabolic block, a change in the nature of -7-the nutrient can induce expression. Furthermore, the activity of a. number of promoters in the glycolytic pathway can be affected by the repression or activation of expression by the GCR gene or other regulatory controls. Also, the GCR regulatory signals can be used to titrate the polypeptide functioning as the regulator for expression of GCR. By having vectors whose copy number can be controlled, one can vary the activity of the wild type GCR gene.

In order to obtain expression, an extrachromosomal element construct will be prepared having a number of sequences defining different functions. One function is the replication system, which forms part of a vector. Another function is a promoter by itself or in conjunc-tion with the alien DNA. Other functions include initiators and terminators of expression. Also, there will be selectable markers.

In developing an appropriate vector, while not necessary, it will be common to have both a replication system for yeast and a replication system for" a prokaryote (a shuttle vector) . The replication system for yeast may be one which provides for stable maintenance of an extrachromosomal element or one which provides a sufficient lifetime for the DNA in the host, that there is an acceptable probability of integration of the DNA into the host. Integration can be greatly aided by providing for a sequence homologous to the host DNA, so as to provide for recombination. Generally, the homologous sequence will be at least about 800bp- usually not more than about 2000bp. Therefore, either integration or an autonomous replication system, such as the use of the ARSl gene, may be employed to provide for the maintenance of the alien DNA in the yeast host. The replication system which is chosen should provide for a reasonable copy number usually greater than 1, pref- -8-erably greater than 5. A wide variety of replication systems are available on a wide variety of prokaryotic vectors, such as pBR322, pACYC184, pSClOl, pMB9, etc. Alternatively, one or more copies of the DNA construct can be integrated into the host chromosome. The replication systems may also be conditionally regulated, usually being temperature sensitive so that replication can be turned on and off by varying the temperature.

In addition to the replication system, there will also be one or more selectable markers, there usually being at least one marker in addition to the alien DNA, which may serve as a marker. Conventional markers include biocidal markers providing antibiotic resistance and those providing resistance to toxins and heavy metal. Also useful is employing an auxotrophic host and providing prototrophy by complementation. In addition to the conventional selection systems just described, the glycolytic genes of the present invention are particularly desirable markers since they can provide for selection, using sugars as selective substrates, in appropriate mutant host strains.

Other genes may also be inserted into the extra-chromosomal element for a variety of purposes. Where integration is desirable in the genome of the host, a homologous sequence for a particular region of the host genome may be included in the extrachromosomal element. Where amplification of one or more sequences is desired, genes known to provide such amplification, such as dihydrofolate reductase genes, which respond to methotrexate stress or metallothionein genes, which respond to heavy metal stress, may be included in the extrachromosomal element, flanked by the DNA regions to be reiterated. Other regulatory signals may also be included, such as centromeres, autonomously replicating segments, etc.

In order to isolate the promoters of interest, clones can be made of yeast chromosomal DNA by random digestion or mechanical shearing of the yeast genome. The presence of the desired gene is then determined by introducing a homogeneous clone of a yeast fragment into an auxotrophic host for complementation. Desirably, the cloning vehicle may have another gene which allows for an additional basis for selection, so that double selection techniques can be used. The mutants are substantially incapable of growing on limited nutrient medium, so that one can select for the presence of the desired glycolytic gene by the choice of medium. After isolating the yeast fragment having the desired gene, the fragment may be subcloned so as to remove superfluous DNA flanking regions and provide for a fragment which is more easily manipulated. The smaller fragment containing the desired gene, of a size less than about 500 base pairs may then be further cloned, restriction mapped and sequenced, so as to provide a useful source for the desired promoters and insertion of the alien DNA. Also, as indicated, the promoters in themselves may be useful, in acting as a titrater for repressor or activator, where it is desirable to modulate the production of a particular enzyme in the yeast host. The alien DNA may be from any source, either naturally occurring or synthetic, either prokaryotic or eukaryo-tic. Of particular interest are mammalian genes which express a poly (amino acid), that is, polypeptide or protein which has physiological activity. To varying degrees, poly (amino acids) prepared in yeast may be modified by glycosylation, where the glycosylation may not occur or may occur at different sites from the naturally occurring mammalian polypeptide and/or in different degrees with different saccharides. It is -10-therefore of great interest to be able to prepare polypeptides which are different from the naturally occurring polypeptide by the degree and manner of glycosylation and in many instances may differ in one or more ways as to the amino acid sequences, where there may be deletions of one or more amino acids or substitutions of one or more amino acids. Mammalian genes may come from a wide variety of mammalian sources, such as domestic animals (e.g. bovine, porcine, ovine and equine) and primates e.g. humans and monkeys.

As exemplary of the use of the subject promoters in preparing an active polypeptide composition, as well as being of particular interest for a variety of purposes, a protease inhibitor is described and made. The protease inhibitor has the same or substantially the same amino acid sequence of human alpha-l-antitrypsin and is capable of inhibiting a number of proteolytic enzymes. The human alpha-l-antitrypsin gene appears to reside within a 9.6 kb EcoRI DNA fragment in the human genome. The mature mRNA appears to have about 1400 nucleotides. One human alpha-l-antitrypsin cDNA has the sequence shown in FIG. IB. The predominant form of human alpha-l-antitrypsin is shown in FIG 1A. Other naturally-occurring forms (polymorphisms) are known.

The sequencing of chromosomal DNA coding for alpha antitrypsin has been described by Kurachi et al., Proc. Natl. Acad. Sci. U.S.A., 78, 6826-6830 (1981) and by Chandra et al., Biochem. Biophys. Res. Comm., 103 , 751-758 (1981), the disclosures of which are incorpo-rated herein by reference. A primate gene for alpha-l-antitrypsin may be obtained by DNA cloning methods described by Chandra et al., ibid. The gene coding for the predominant form of human alpha-l-antitrypsin, isolated from a human cDNA library by using the baboon sequence as a DNA hybridization probe is shown in FIG. 1A.

The human alpha-l-antitrypsin has a BamHI restriction site which allows the cutting of the gene with the removal of information for a single glutamic acid from the mature protein. Various schemes can be employed for introducing the human alpha-l-antitrypsin gene adjacent the glycolytic promoter to be under the regulation of the promoter. Where the promoter does not have a convenient restriction site near the f-met codon, the glycolytic gene may be cleaved and chewed back to the promoter with Bal31. A linker may then be introduced downstream from the promoter to provide a convenient cohesive end or flush end for joining to the human alpha-l-antitrypsin gene. The linker can also provide one or more codons for amino acids at the N-terminus of the alpha-l-antitrypsin gene, which may be the same or different from the naturally occurring amino acids.

The gene for human alpha-l-antitrypsin may then be inserted into the extrachromosomal element downstream from the glycolytic promoter, where an f-met codon is provided for initiation of expression of the human alpha-l-antitrypsin.

For example, the cDNA coding for alpha-l-antitrypsin (hereinafter "AT") may then be inserted into an expression vector, such as CTEA32 (FIG. 2) , which contains the yeast promoter for triose phosphate isomerase (TPI) inserted at the BamHI site of the shuttle plasmid, CV13 [Broach J.R. , Strathern J.N., Hicks J.B., Gene, 8^: 121-133 (1979)]. A synthetic DNA adaptor was ligated into the TPI promoter after the TPI structural sequences were removed by BAL31 digestion from the Kpnl restriction site within the TPI coding region. (Alber et al, J. Molec. Applied Genet., 1, -12- 419-434 (1982)). This adaptor contained an ATG codon for; translation initiation, followed by the sequence GAGGATCC. The GAG codon specifies a glutamic acid residue, which is the first amino acid of the natural-ly-occurring human AT. The GGATCC portion of the adaptor is a cutting site for BamHI endonuclease and allows for the splicing of the remainder of human AT DNA sequence into this vector.

The BamHI site of CTEA32 was constructed to be "in frame" with the rest of the AT structural gene, thereby allowing for the expression of the polypeptide when a BamHI fragment from the cloned cDNA is appropriately inserted into CTEA32. The plasmid consisting of CTEA32 plus the AT gene is called CAT1 (FIG. 2) .

This DNA construct containing the gene for human AT located downstream to a yeast triose phosphate isomerase (TPI) promoter fragment was transformed into yeast strains, N501-B and GK100. Transformation into yeast is described by Beggs, Nature, 275, 104-109 (1978). Screening of the transformed yeast strains by immunological assays (competition assays and ELISA assays, using antibodies against alpha-l-antitrypsin) confirmed the presence of large amounts of human AT in yeast made from the plasmid CAT1. The "wild-type" yeast strain, N501-1B (described by Kawasaki et al., Biochem. Biophys. Res. Comm., 108, 1107-1112 (1982)), when transformed with CAT1, produced 1.8 mg alpha-l-antitrypsin per gram of soluble protein (or 0.18% alpha-l-antitrypsin), when grown at 30° on a synthetic minimal medium (modified Wickerham's medium) with 6% glucose. A mutant yeast strain, GK100, when transformed with CATl, produced 10-15 mg alpha-l-antitrypsin per gram soluble protein (or 1-1.5% alpha-l-antitrypsin) under the same growth conditions. Strains N501-1B and GK100 each carry a defective LEU2 gene which allows for the selective -13-maintenance on minimal and leucine-less media of CV13 and CV13-derived plasmids (such as CAT1) which each contain a functional LEU2 gene. When grown on minimal media with only CV13 as a control, N501-1B and GKIOO produce no detectable AT. Thus, AT may be specifically produced by the CAT1 plasmid.

Since GKIOO produces significantly more AT than N501-1B, it is preferred. However, the present invention is not limited to AT production by GKIOO. It may be desirable to utilize mutations in GK100 which lead to hyperproduc-tion of AT.

An immuno-adsorption column, made according to the method of Cuatrecasas, P. J. Biol. Chem. , 245, 3059 (1970), was prepared by covalently attaching affinity-purified goat antibodies to human AT to CNBr-activated Sepharose. Disrupted GK100 yeast cells were extracted with 3 volumes of phosphate buffered saline pH 7.2 containing 0.5M NaCl, and the extracts were applied to the column. Yeast produced human AT (0.5-1.0 mg) was eluted from the column with 3M NaSCN. After the material was dialyzed to remove salt it was analyzed by electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate, the results of which are shown in FIG. 3. Based on the relative migration of the protein in the gel, the approximate molecular weight of the human alpha-l-antitrypsin made in yeast is 42,000- 43,000 daltons. Naturally occurring human AT has a molecular weight of approximately 54,000 daltons, having a carbohydrate composition of approximately .16% by weight, as shown by Hodges et al, J. Biol . Chem. , 254 , 8208-8212 (1979) . It therefore appears that the yeast produced AT may be unglycosylated or substantially unglycosylated and may lack carbohydrate portions present in the naturally occurring protein.

Alternatively, other expression vectors may be constructed which contain a segment coding for alpha-1-antitrypsin. Such expression vectors may be constructed by methods known to those of ordinary skill in the art using available DNA constructs. A preferred vector is plasmid Cl/1, which is more stable than CV13 and CV13 derived vectors, such as CATl. Cl/1 was constructed from plasmid, pJDB248 (Beggs, J., Nature , 275, 104-109 (1978)). The pMB9 sequences were removed from pJDB248 by partial digestion with Eco RI and were replaced by pBR322 DNA which was cut with Eco RI. The restriction map of Cl/1 is given in FIG.. 4. The Cl/1 plasmid contains the entire 2-micron DNA from yeast (S . cer-visiae) , with a pBR322 insertion at an EcoRI site. It also contains the LEU2 gene. Thus, the yeast TPI pro.motor with the adaptor may be inserted into the single BamHI site in the Tc gene of Cl/1. Then the AT sequence, attached to a transcription terminator fragment from the yeast TPI gene, may be inserted into the BamHI side downstream from the TPI promotor. The resulting plasmid, HAT4 , may then be transformed into N501-1B and GK100 in a manner as described above.

The sequence of the first ten amino acids in the yeast-produced AT may be confirmed by amino acid sequence analysis as identical to the first ten amino acids of the naturally occurring human AT: H2N-Glu-Asp-Pro-Gln-Gly-Asp-Ala-Ala-Gln-Lys°- The yeast-produced AT does not contain the initiation methionine which is specified by the ATG start codon. Therefore, the yeast cell processes off the methionine to produce the amino acid sequence of natural human AT.

The polypeptides produced according to the present invention having AT activity may be useful for treatment -15-of a genetic AT deficiency and other diseased states related to inadequate levels of AT. Thus, conditions such as emphysema and other lung disorders related to progressive digestion of lung sacs may be treated, such as, chronic obstructive pulmonary disease or adult respiratory distress syndrome. Non-genetically related emphysema may also be treated, such as, emphysema resulting from heavy smoking. Conditions not necessarily confined to the lungs may also be treated, such as, cystic fibrosis and arthritis. For a review of AT deficiency, see Gadek, J.E. , and R. Crystal, "Alpha-1-Antitrypsin Deficiency", The Metabolic Basis of Inherited Disease, J.B. Stanbury, J.B. Wyngaarden, D.S. Fredrickson, McGraw-Hill, N.Y. pp. 1450-67 (1982).

The alpha-l-antitrypsin can be used as an antigen for production of polyclonal and monoclonal antibodies to human alpha-l-antitrypsin, for introduction into a host having a deficiency of alpha-l-antitrypsin, or for modulating proteolytic activity in a mammalian host. In Partikular, the alpha-l-antitrypsin can be administered to humans to replace alpha-l-antitrypsin which has been inactivated (oxidized) by tobacco and other smoke.

The polypeptides according to the present invention may be admixed with conventional pharmaceutical carriers. Preferably , the polypeptides are to be administered intravenously or by inhalation. While the effective dosages may vary according to the severity of the condition and weight of the subject, dosages in the range of 0.5-10.0 gm/week of a polypeptide introduced intravenously may, in many cases, be effective. Lower dosages may be effective if the method of administration is by inhalation. Oral administration may also be effective provided the AT is protected in capsules or coated carriers from premature degradation in the digestive tract.

The following examples set forth specific embodiments according to the present invention, but the invention is not intended to be limited thereto.

EXAMPLE 1 Strains. Isogenic strains carrying mutations in PGI1 , PGK1 , GPM1 , PYK1, and GCR1 where obtained by ethyl methane sulfonate (EMS) mutagenesis of S. cerevisiae (S. c.) X2180-1A (MATa SUC2 CU l ga!2, from the Berkeley Yeast Stock Center). 35,000 independent colonies were, grown on YEP-3% glycerol-2% ethanol and were screened by replica plating for the inability to grow on YEP-4% dextrose (Table 1).

Identification of specific lesions was made by comple-- mentation tests with known glycolysis mutants (Ciriacy and Breitenbach, J.Bacteriol , 139; 152-60 (1979)), while at least 15 additional complementation groups were found by intercrossing mutant strains. Enzyme assays (Clifton et al. Genetics, ^8:1-11 (1980)) confirmed the glycolytic defects in pgil , pgkl , gpml, pykl, and gcrl mutants.

A LEU2 mutant was also derived from S. cerevisiae X2180-1A by EMS treatment and was crossed to X2180-1B (an isogenic MATa strain) to produce N501-1B (MATa leu2 SUC2 CUPl ga!2) . Cycloheximide (cyh2) and canavanine (canl) resistances were then selected as spontaneous mutations in N501-1B. The glycolysis mutants were crossed to N501-1B to produce a series of isogenic leu2 strains each defective in a single glycolytic function or in GCR1.

A tpil mutant, S. cerevisiae GLU77 was crossed to N551-1A (MATa leu2 SUC2 CUPl ga!2) ; strains derived from this mating were crossed twice to N501-1B to produce a -17-tpil leu2 strain, N587-2D, which was similar in genetic background to the other Glycolysis mutants.

Mutations in three glucose phosphorylating enzymes produce a strain which is unable to grow on dextrose as the sole carbon source and which is resistant to catabolite repression by 2-deoxyglucose and glucosamine. N517-6C (hxkl hxk2 qlkl leu2 canl-100 cyh2 ade2-l) was derived from a hxkl hxk2 glkl strains, D308.3, by screening for glucosamine- resistant spore colonies. Defects in glucose kinasing activities were confirmed by assay. -18- TABLE 1 Complementation Groups of glu" Derivatives of X2180-1A Gene No. of Mutants PYK1 14 PDC1 9 GCR1 4 PGI1 3 GPM1 3 PGK1 1 TPI1 0 FDP 0 (LEU2) (1) I 11 II 10 III 3 IV 5 V 1 VI 1 VII 2 VIII 3 60 other mutations not in IX 2 the complementation groups X 3 XI 2 XII 1 XIII 1 XIV 5 XV 1 27 sterile glu strains 35,000 colonies screened (EMS mutagenized for 50% kill) -19- The homothallic diploid strain, j3. c. AB320 was the source of the yeast DNA pool {Nasmyth and Reed, Proc. Nat. Acad. Sci., 77:2119-2123 (1980) and was used as a control in some experiments.

The triose phosphate isomerase gene (including the upstream sequence having the regulatory signals) is as follows : t( C>Ttllltl')ICI1 c c i r.tc liCCCT«(tl(CCltlt1TT iiiittiTTtiicirtcrtioiiiitcccittr -Ma -β» -t» -«· -B» -Μ» Tttl TI CTITCXIiCCITIM tTciiicictt oiici . T 1 net cccticiittrcccrTCiictitiiitcriii -*m -aa cce irC tct CTttt»c»teectttt« T.I > C 4 T CCIirttlfUtCtt(lltllCM4lllClttCt -ai -Mi -a. -Bi -11·· -a cc« llllTlltTitllClll C t 1 ■ et IITCTCTCI TlCClCCfCllttCIIICTIKI - -aa -mm -«■ «.r (CTirirrtCiortci t * 1 r. c * ■ iiiticcriTirxcrrrttcrccccriciiri -a. -xa -ua -aa ' -im .im tet tTC ccitetf ncitiiitu TI net ttttiittttiiiteiiKTtitrei ititii -ut -*a ~m S -m -a -m • ititrcttttcrticfi * - 1 i T e T ITllCllCUIIIlOClTIClltllCtlltllT ·" -m -» -a -a -it -i t etc IllllCIIICTIIIftl . T 1 lift icitniiTliiicliniciiiciiicciitii U a a a a t C 1 ICTI cf tcttTCCiKiiitti!ciiiiTtcititcrt 1· B » ia ua »cc TtCICCf t CtlCtTK * C 1 tier ut ia ua a* i½ ua c. ticrciiiiciccttci T( 1 111. cttcticticmcicciitiintttcciTiii im m aa a. at ia «i ■ at -a t at, «1 ts Br- aL t-C u cu ca aa aa ac ac aB Ta llt llillttlttlTI C T 1iiiriiciiiiitiiiciuctcciiitcticititTt a. a* m m m CTT cctectititiiCiKi T C J T T . C TttCttttCCttttTCCTTTtCCTCttCCTCT lU. Bi a» at at tm c«. TtTtltCTtlTltlTII . T 1 taETtriiiKuiiliiiictcctacicttf cgi Π» Mi a» aa at tm T.t T »«r ttciticciicoitci * rtc.Ttcc.tTTTt.c aa aa .. « C C T.CTtcTcctcttctT.c Ut-— UC Ut Mt Tet ItlTlItClIlCttCCI T c 1 S t t tCTTCTT.CCTf CCttCTT.tCTCtCt-CCCTCC M* M* •a at aa ta etc cttirritditctTii * e 1 criirrcccttiicECTXtiicitcctticcTTcit U* ca oa M. tM «· ...tllKKIllltClltl . T 1 T C T tiiTCCcTiiTiitictitiiKicxiiritit ca M aa at Tie ra T * T Cm Ml «* •a *a .a TCT TtilHttlltltTITI T * 1 iir tttTT.tr. cttc.ttt.cTi TTTttTtTT.T .« *a ^a ^ TT« TTTTTC»TtcTct.cca C 11 rmt rtTC.T«ttt.TTCTT.Ttt...tc..T*.tTTT •ua The transformants were purified on leucineless media and were then grown on a non-selective medium (YEPGE) to allow mitotic segregation of the plasmids. Strains which cosegregated the leu2 and glycolysis mutant phenotypes, as determined by replica plating on selective media, were assayed for glycolytic enzyme activities. Yeast DNA preps were made, and the E. coli strain, RR1, was transformed, selecting for ampicillin resistance, to verify the presence of plasmid DNAs in these yeast glycolytic transformants.

Enzyme Assays. The transformed yeast strains were selectively grown on minimal medium with 8% glucose (adenine was added to a final concentration of 50mg/l) . The wild-type control, N501-1B, was grown on the same medium plus leucine (lOOmg/1) . The glycolysis mutant strains were grown on YEP-5% glycerol-1% . lactate. Overnight cultures were fed fresh media and were aerobically grown at 30° for four hours before harvesting. The cells were washed two times with water and resuspended in 50mM K2HP04 2mM EDTA 3mM 2-mercapto-ethanol (adjusted to pH7.4 with HCl) . Extracts were obtained by vortexing the cells with an equal volume of glass beads (.45 mm diam.) at high speed for two minutes. The cell debris was removed by centrifugation in a microfuge for 15 min. at 4°. Enzymes were assayed as described by Clifton and Breitenbach, supra. Protein concentrations were determined by the Biuret-TCA method.

EXAMPLE 2 In order to determine the activity of the various glycolytic genes in the transformants, the various enzymes were assayed and the results for. the transformants were compared to mutant and wild-type strains. The gcrl mutant had 5-10% of the wild-type levels of most glycolytic activities (exemplified by PGI, aldolase and enolase) and grows very poorly on glucose media. In contrast, the GCR1 transformants had nearly wild-type levels of enzymes and were virtually identical to wild-type for growth on glucose media. The other glycolysis mutants had less than 5% of the normal levels of their respective enzyme activities. However, when transformed with a complementing high copy plasmid, the specific enzyme activities were substantially elevated above wild-type levels (typically 5-10 fold higher) . The following Table 2 indicates the results. -23- ABLE 2 Comparison of Glycolytic Activities in Wild-type Mutant, and Transformed Strains zyme Activities Ratio: Wild-typea Mutant5 Transformant Transf/Wt PGI 2.85 .0065 31.49 (10) 11.1 TPI 18.3 .0000 167.8 (10) 9.2 PGK 1.99 .0046 17.67 (3) 8.9 GPM 0.74 .0000 4.80 (10) 6.5 PYK 4.02 .0057 14.77 (10) 3.7 Wild-type3 gcrl Mutant GCR1 Transf'c PGI 2.85 .2436 2.42 (10) .85 Aldolase 4.33 .4415 2.96 (10) .68 Enolase 0.43 .0274 .316 (10) .74 aWild-type is N501-1B.

^The respective mutant strains are N543-9D (pgil leu2) , N587-2D (tpil leu2) , N548-8A (pkgl leu2) , N583-2C (gpml leu2) , and N549-3A (pykl leu2) .

°The activities of the transformants are averages for many different isolates. The numbers in parentheses represent the numbers of independent transformants assayed. dThe gcrl leu2 mutant strain is N525-2C.

EXAMPLE 3 In order to demonstrate that the hyperproduction of glycolytic enzymes was specific to the mutational defect complemented by the particular plasmid, assays for ten -24-different glycolytic proteins were conducted on the various transformants . The following Table 3 reports the results for one transformant for each of the six different glycolysis genes which were examined in detail .

TABLE 3 RELATIVE ENZYME ACTIVITIES OF WILD-TYPE AND TRANSFO GLYCOLYTIC ENZYMES Strains GLK PGI PFK FBA TPI GLD N501-1B 1.00 1.00 1.00 1.00 1.00 1.00 Transformant GCR-8 1.05 0.63 1.44 0.79 0.62 0.63 Transformant PGI-19 0.64 5.63 1.26 0.57 0.58 0.75 Transformant TPI-10 0.99 0.77 1.35 0.99 13.85 0.87 Transformant PGK-2 0.54 0.45 1.05 0.54 0.46 0.63 Transformant GPM-2 0.97 0.82 1.69 1.02 1.02 0.85 Transformant PYK-1 1.02 0.83 1.09 0.89 1.22 0.84 -26- The GCR-8 transformant gave nearly wild-type levels of all ten enzymes, while PGI-19, TPI-10, PGK-2, GPM-2 and PYK-1 transformants overproduced their , respective glycolytic proteins, but not other enzymes. it was noted that the plasmids readily segregated (typically 5-50% segregation in fully grown cultures even under selective pressure of leucine prototrophy, so the assayed cultures probably contain cells with a range of number of plasmids. By complementation in E. coli and/or sequencing, TPI1 and PYK1 have both been shown to be the structural gene.

EXAMPLE 4 Exploitation of the promoter for TPI1 for the production of human alpha-l-antitrypsin was demonstrated as fol-lows. The plasmid CV13 was employed. CV13 can be maintained by selection of yeast with an average of about ten copies per cell. CV13 is comprised of pBR322, the replicon for the 2y-plasmid and the yeast LEU2 gene. TPI1 promoter fragment was obtained by cutting the TPI1 gene at the unique Kpnl site (bases 511 to 518); and the resulting linearized DNA was then treated with Bal31 for four to five minutes in order to remove the TJPI1 structural sequences. Linkers, either EcoRI, Hind III or BamHI, were then inserted. The linkers will then cleave with the appropriate Restriction enzyme to provide cohesive ends for insertion of human alpha-l-antitrypsin genes. The human alpha-l-antitrypsin gene was digested with BamHI, which cleaves at the 5 '-terminus of the coding strand to remove the information for a single glutamic acid codon from the mature protein. Four different constructions were prepared, as set forth in the following Table 4. From this table it is noted that the glutamic acid codon is substituted by the codons for alanine and proline in three of the constructions having the initiator methionine.

After ligation of the human alpha-l-antitrypsin construction into the CV13 plasmid, the resulting plasmid was transformed into S. c. N501-1B. The resulting yeast cells were then grown on a minimal synthetic medium.

TABLE 4 Plasmid N-terminal amino acid Orientation in CV13 CA 1 met glu + hAT* clockwise C-Tct2 met ala pro + hAT counterclockwis C-Tcxl met ala pro + hAT clockwise C-TSct2 met ala pro + hAT, but counterclockwis missing part of TPI promoter *remainder of approximately 400 amino acids of human alpha-l-antitrypsin Yeast cells containing the human alpha-l-antitrypsin genes were broken open by vortexing with glass beads (0.45mm) at high speed for 2-3 minutes. The extraction buffer contained 50mM K2HP04, 2mM EDTA, 2mM 2-mercapto-ethanol and lmM P SF (pH7.4)icell debris was removed by centrifugation and the extracts contain 3-4mg/ml protein as determined by Lowry assays.

The presence of human alpha-l-antitrypsin was determined using a RIA, employing tritium-labeled human alpha-l-antitrypsin and antibody directed against the protein. The following Table 5 indicates the results. -28- TABLE 5 Competition assay for alpha-1 antitrypsin Tritium Average α-1-ΑΤ Total %Total Plasmid Counts Count [ug] Protein (yg) Protein CAT1 46010 49133.5 0.75 420 .18 52257 C-Ta2 12268 12799 3.35 380 .88 13330 C+Tal 41635 40353 0.95 360 .26 39071 C-TSa2 66490 68264 0 345 0 70038 Controls** Counts 0 yg a-1 68440 0.25 yg a-1 65333 0.5 yg a-1 58928 1.0 yg a-1 38468 2.0 yg a-1 19559 1 3.0 yg a-1 14432 4.0 yg a-1 11155 5.0 yg a-1 9615 *Plasmids were grown in yeast strain, N501-1B. lOOyl of extracts were assayed.

**Non-radioactive alpha-1 antitypsin mixed with lOOyl of yeast extract (330yg protein) It is evident from the above results that an immunologically active product is obtained, which is capable of competing with naturally occurring human alpha-1-antitrypsin for antibodies to the native protein. -29- Furthermore, the expression of the alpha-l-antitrypsin gene is regulated by the TPI promoter, for as is seen, where a portion of the TPI promoter is removed, no alpha-l-antitrypsin is produced. In addition, the production of the mammalian protein human alpha-l-antitrypsin has not been optimized in the above study, so that the results indicate a minimum production of product which can be further enhanced. Thus, the TPI promoter is found to be an effective promoter for efficiently producing high yields of expression products of alien DNA.

EXAMPLE 5 Purification Of Alpha-1-Antitrypsin From Yeast GK100 Yeast Extracts An immuno adsorption column was prepared by covalently attaching affinity-purified antibodies to human alpha-l-antitrypsin to CNBr-activated Sepharose according to the method of Cuatrecasas, J. Biol. Chem. , 245 , 3059 (1970). Disrupted GK100 cells were extracted with three volumes of phosphate buffered saline pH 7.2 containing 0.5M NaCl and applied to the column. The column was eluted with 3M NaSCN and the recovered material was analyzed by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. The results of the electro-phoresis are shown in FIG. 3. Track 1 contained a mixture of molecular weight standards: a) phosphorylase B, 97,000 daltons; b) bovine serum albumen (BSA) , 65,000 daltons; c) ovalbumin, 43,500 daltons; d) carbonic anhydrase, 30,000 daltons; e) soybean trypsin inhibitor, 20,000 daltons; and f) alpha-lactalbumin, 14,000 daltons. Track 3 contains yeast produced AT purified by immunoadsorption, molecular weight about 42,000 daltons. Track 7 is a sample of naturally occurring AT purchased from Sigma Chemical Company, heavily contaminated by -30-blood proteins. A major component of Track 7 is human alpha-l-antitrypsin, molecular weight 54,000 daltons.

EXAMPLE 6 Activity Of Yeast Produced Alpha-l-Antitrypsin Against Serine Protease Trypsin As a control, 10 microliters (1 microgram) of a solution of 100 microgram/ml trypsin, 100 microgram (100 microliters) of bovine serum albumin and 100 microliters of 0.05 molar TRIS, pH 8.0 buffer containing ImM benzoyl-arginioyl-p-nitroanilide were mixed, and the increase in absorbance at 405 nm was measured over time in a spectrophotometer. The absorbance value of this solution was used as a standard for 100% trypsin activity. Three additional samples were run in duplicate, each containing 1 yl trypsin and, respectively containing 25 yl AT solution plus 175 yl buffer, 100 yl alpha-l-antitrypsin plus 100 yl buffer, and 200 yl alpha-l-antitrypsin. All samples contain equal concentrations of substrate and bovine serum albumin. The results demonstrated that utilizing 25 microliters of AT, 73% of the trypsin activity remained, with 100 microliters of AT, 41% of trypsin activity remained and with 200 microliters of alpha-l-antitrypsin, 26% of the trypsin activity remained. This demonstrated that by increasing the levels of the yeast made AT the trypsin inhibitory activity also increased.

EXAMPLE 7 Production of Alpha-l-Antitrypsin From Yeast Plasmids With Increased Genetic Stability Increased levels of AT may be obtained by utilizing Cl/1, a plasmid which is more genetically stable than -3.1- CV13. The Cl/1 plasmid contains the entire 2-micron DNA from S_;_ cerevisiae and, therefore, can promote its own replication and maintenance in yeast in the absence of selection for a genetic marker. Also, Cl/1 plasmid has R a single BamHI site located in the Tc gene. Trans-formants carrying Cl/1 may be selected in coli by ampicillin- or tetracycline-resistance and in yeast by leucine prototrophy. Cl/1 contains pBR322 inserted into an EcoRI site of 2-micron DNA and carries the LEU2 gene described by J. Beggs, Nature, 275, 104-109 (1978).

The yeast TPI promoter (from CTEA32) with the synthetic DNA adaptor (described above) was inserted as a Bgl II -BamHI fragment (about 900 base pairs) into the BamHI site of Cl/1. This insertion created a single BamHI site into which the human AT gene could be spliced for expression in yeast. As in the CAT1 plasmid, when the AT gene (FIG. 1A) is inserted, the resultant plasmid would have an ATG initiation codon followed by a GAG (glutamic acid codon) to allow the production of mature human AT protein sequence in yeast.

About 700 base pairs of the 3* flanking region of the yeast TPI gene was added after the human AT sequence to assist in transcription termination. The "termination" fragments are sequences from the Xbal to EcoRI sites in the plasmid TPIC10 (T. Alber and G. Kawasaki, J. Molec. Applied Genet. , 1, 419-434 (1982)).

The yeast termination sequences were attached to the human AT gene by using the vector, pUC13, which has multiple cloning sites into which the terminator and AT DNA' s can be separately inserted. The pUC13 plasmid was constructed as described in Vieira, J., and Messing, J., Gene, 19, 259-268 (1982) for vectors, pUC8 and pUC9. The pUC13 plasmid contained the multiple restriction -32-site, depicted in FIG. 5, at the start of the lac Z gene. To connect the human AT gene to the TPI transcription terminator, the AT cDNA clone (FIG. 1) was inserted as a Pst I fragment into pUC13 at the single Pst I site. The AT gene was followed by an Xba I site and Eco RI site in the multiple cloning sequence. Between these Xba I and Eco RI sites of pUC13 was inserted the yeast TPI terminator as a 700 base pair Xba I-Eco RI fragment from pTPIClO. The resulting plasmid, pUCcxl+FGl, contained a human AT gene with a yeast transcription terminator (See FIG. 6) . An Eco RI-Bam HI synthetic DNA adaptor was then added to the Eco RI site of the plasmid, in order to create a Bam HI site on the 5' end of the yeast terminator. By using this adaptor, the human AT-yeast terminator sequence could be removed by cutting with Bam HI to liberate a fragment of approximately 2100 base pairs. This BamHI fragment was inserted into the Cl/1 plasmid containing the TPI promoter with BamHI adaptor. The resulting plasmid, HAT4, has the TPI promoter, ATGGAGGATCC adapter, human AT gene (from the BamHI site) , and TPI terminator inserted into Cl/1. The topology of HAT4 is depicted in FIG. 7.

HAT4 was transformed into N501-1B and GK100. On minimal media with 6% glucose, 2-3% of the yeast soluble protein was alpha- 1-antitrypsin at a cell density of nearly 3g per liter (wet weight). Because HAT4 contained Cl/1, this plasmid was maintainable in a variety of rich media, including YEPD (1% yeast extract, 2% peptone, and 2% glucose) . On rich media 2-3% AT was still produced but at a higher cell density of 10-20g per liter (wet weight) . The HAT4 plasmid was maintained without selection in N501-1B for over 30 divisions on rich media with greater than 70% of the cells containing the -33-plasmid. In GK100 better than 95% of the cells had HAT4 after 30 divisions on rich media. The advantages of using HAT4 over CAT1 were 1) greater piasmid stability, 2) higher levels of AT as a percentage of total protein, 3) much greater yields of cells per liter as a result of using rich media, and 4) cheaper costs of rich media compared to synthetic (leucine-less) media. The mutant yeast strain GK100 has been placed on deposit in the American Type Culture Collection, Rockville, Maryland, ATCC No. 20669.

It is evident from the above results that yeast promoters can be efficiently used for the production of foreign proteins by regulating the expression of alien DNA in yeast. The promoters are found to be strong promoters, so as to provide for a high degree of expression. Furthermore, it would appear that the messengers are sufficiently stable as to allow for a significant degree of translation into the desired expression product. Furthermore, by employing the glycolytic promoters and appropriate nutrient media, the expression of the alien DNA can be modulated. In this way, production of the alien DNA can be turned on and off. Thus, the subject invention provides a method for using yeast as efficient host in the production of foreign proteins, where the production may be modulated. In addition, by using the glycolytic regulation gene, one can turn on and off a plurality of glycolytic promoters.

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 modification may be practiced within the scope of the appended claims.

Claims (13)

92734/2 - 34 - WHAT IS CLAIMED IS:

1. A method of producing a polypeptide having the protease inhibition activity of human mammalian alpha-1-antitrypsin, comprising the step of growing a yeast culture transformed by a DNA expression vector, said vector containing a promoter which regulates the transcription of a glycolytic protein, which protein is triose phosphate isomerase or pyruvate kinase, said promoter being followed downstream by a gene under the regulation of said promoter comprising a segment coding for human mammalian alpha-1-antitrypsin.

2. A method according to Claim 1 wherein said yeast comprises Saccharomyces cerevisiae.

3. A method according to Claim 2 wherein said Saccharomyces cerevisiae comprise cells of the mutant strain GK-100 ATCC 20669.

4. A method according to Claim 1 wherein said Saccharomyces cerevisiae contains allelic mutations to GK-100 ATCC 20669 whereby hyperproductlon of said polypeptide is attained.

5. A method according to Claim 1 wherein said vector comprises plasmld CAT1.

6. A method according to Claim 1 wherein said vector includes 2-micron plasmid DNA.

7. A method according to Claim 1 wherein said vector comprises plasmld HAT4.

8. A method according to Claim 1 further comprising the steps of extracting said polypeptide from said culture and purifying said polypeptide.

9. A method according to any of Claims 1 to 8 wherein said polypeptide comprises the amino acid sequence of naturally-occurring human mamallan alpha-l-antitryps1n. - 35 - 92734/5

10. A method according to claim 9, wherein said polypeptide is the predominant human form of alpha-l-antitrypsin as hereinbefore defined.

11. A polypeptide produced according to the method of any one of claims 1 to 8.

12. Substantially pure, substantially unglycosylated, active, human mammalian alpha-l-antitrypsin.

13. A substantially pure, substantially unglycosylated, active, human mammalian alpha-l-antitrypsin of claim 12, which is characterized by the amino acid sequence of human mammalian alpha-l-antitrypsin as hereinbefore defined. for the Applicant: WOLFF, BREGMAN AND GOLLER