Method of expressing alpha-1-antitrypsin in yeast
This application is related to Ser. No. 408,099, filed Aug. 13, 1982, now allowed U.S. Pat. No. 4,599,311 the disclosure of which is incorporated herein by reference in its entirety. 
     The present invention is directed to a method of expressing human alpha-1-antitrypsin in yeast and to the protein product expressed thereby. 
     Alpha-1-antitrypsin is a protease inhibitor present in mammalian blood whose apparently major physiological function is to inhibit elastase, a potent protease which hydrolyzes structural proteins. Alpha-1-antitrypsin also inhibits other serine proteases. The normal plasma level of alpha-1-antitrypsin is about 2 mg/ml. A low level of alpha-1-antitrypsin in the blood may be associated with chronic obstructive pulmonary emphysema and infantile liver cirrhosis. Under many inflammatory conditions, an acute-phase response is initiated and the concentration of alpha-1-antitrypsion is substantially increased. In order to study and treat alpha-1-antitrypsin deficiency and to examine the mechanism of the acute-phase response, it is therefore desirable to have a pure alpha-1-antitrypsin protein. In particular, it is desirable to have a source of antitrypsin protein produced by microorganisms through genetic engineering techniques. 
     It is therefore an object of the present invention to provide a method for producing alpha-1-antitrypsin in microorganisms, preferably in yeast. 
     It is a further object of the present invention to provide a pure alpha-1-antitrypsin polypeptide which has been profuced by genetic engineering techniques. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     In the accompanying FIGURES: 
     FIG. 1 is the cDNA sequence of the gene coding the predominant form of human alpha-1-antitrypsin. 
     FIG. 2 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 C1/1. 
     FIG. 5 illustrates the DNA sequence of the multiple restriction site of pUC13. 
     FIG. 6 illustrates the restriction map of plasmid pUCα1 containing the DNA sequence from FIG. 1. 
     FIG. 7 is the restriction map of plasmid HAT4. 
    
    
     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 incorporated herein by reference. A primate gene for alpha-1-antitrypsin may be obtained by DNA cloning methods described by Chandra et al., ibid. The gene coding for the predominant form of human alpha-1-antitrypsin, isolated from a human cDNA library by using the baboon sequence as a DNA hybridization probe is shown in FIG. 1. 
     The cDNA coding for alpha-1-antitrypsin (hereinafter &#34;AT&#34;) 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 KpnI restriction site within the TPI coding region. (Alber et al, J. Molec. Applied Genet., 1, 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 naturally-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 &#34;in frame&#34; 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-1-antitrypsin) confirmed the presence of large amounts of human AT in yeast made from the plasmid CAT1. The &#34;wild-type&#34; 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-1-antitrypsin per gram of soluble protein (or 0.18% alpha-1-antitrypsin), when grown at 30° on a synthetic minimal medium (modified Wickerham&#39;s medium) with 6% glucose. A mutant yeast strain, GK100, when transformed with CAT1, produced 10-15 mg alpha-1-antitrypsin per gram soluble protein (or 1-1.5% alpha-1-antitrypsin) under the same growth conditions. Strains N501-1B and GK100 each carry a defective LEU2 gene which allows for the selective 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 GK100 produce no detectable AT. Thus, AT may be specifically produced by the CAT1 plasmid. 
     Since GK100 produces significantly more AT than N501-1B, it is preferred. However, the present invention is not limited to AT production by GK100. It may be desirable to utilize mutations in GK100 which lead to hyperproduction 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 affinitypurified 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-1-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 C1/1, which is more stable than CV13 and CV13 derived vectors, such as CAT1. C1/1 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 C1/1 is given in FIG. 4. The C1/1 plasmid contains the entire 2-micron DNA from yeast (S.cervisiae), with a pBR322 insertion at an EcoRI site. It also contains the LEU2 gene. Thus, the yeast TPI promotor with the adaptor may be inserted into the single BamHI site in the Tc R  gene of C1/1. 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: ##STR1## The yeast-produced AT does not contain the initiation methionine which is specified by the ATG start codon. 
     The polypeptides produced according to the present invention having AT activity may be useful for treatment 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, &#34;α1-Antitrypsin Deficiency&#34;, The Metabolic Basis of Inherited Disease, J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, McGraw-Hill, N.Y. pp. 1450-67 (1982). 
     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 
     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-1-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 electrophoresis 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 blood proteins. A major component of Track 7 is human alpha-1-antitrypsin, molecular weight 54,000 daltons. 
     EXAMPLE 2 
     Activity Of Yeast Produced Alpha-1-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 1 mM benzoylarginioyl-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 μl trypsin and, respectively containing 25 μl AT solution plus 175 μl buffer, 100 μl alpha-1-antitrypsin plus 100 μl buffer, and 200 μl alpha-1-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-1-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 3 
     Production of Alpha-1-Antitrypsin From Yeast Plasmids With Increased Genetic Stability 
     Increased levels of AT may be obtained by utilizing C1/1, a plasmid which is more genetically stable than CV13. The C1/1 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, C1/1 plasmid has a single BamHI site located in the Tc R  gene. Transformants carrying C1/1 may be selected in E. coli by ampicillin- or tetracycline-resistance and in yeast by leucine prototrophy. C1/1 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 Bg1 II BamHI fragment (about 900 base pairs) into the BamHI site of C1/1. 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. 1) 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&#39; flanking region of the yeast TPI gene was added after the human AT sequence to assist in transcription termination. The &#34;termination&#34; fragments are sequences from the XbaI 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&#39;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 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 pTPIC10. The resulting plasmid, pUCα1+FG1, 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&#39; 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 C1/1 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 C1/1. 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 3 g per liter (wet weight). Because HAT4 contained C1/1, 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 10-20 g 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 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 plasmid 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.