WO1991018988A1

WO1991018988A1 - Barrier protease

Info

Publication number: WO1991018988A1
Application number: PCT/US1991/003952
Authority: WO
Inventors: Vivian L. Mackay; John W. Forstrom
Original assignee: Zymogenetics, Inc.
Priority date: 1990-06-08
Filing date: 1991-06-05
Publication date: 1991-12-12
Also published as: AU8106091A

Abstract

Compositions of barrier protease are provided, as are methods of producing barrier protease from cell culture media. The barrier protease compositions may be used within a variety of industrial processes, particularly those calling for the cleavage of leucine-lysine or leucine-arginine bonds under acidic conditions.

Description

BARRIER PROTEASE

Technical Field

The present invention relates to proteolytic enzymes and their preparation. More specifically, the present invention relates to barrier protease, methods of preparing barrier protease, and compositions containing barrier protease.

Background of the Invention

Enzymes have a broad range of industrial applications in such fields as food processing, wine making, brewing, textiles, leather processing and pulp and paper manufacturing. Enzymes are also important components of laundry detergents and other products. Through the utilization of enzymes, industrial processes can be carried out at reduced temperatures and pressures and with less dependence on the use of corrosive or toxic substances. The use of enzymes can thus reduce production costs, energy consumption and pollution as compared to non-enzymatic products and processes.

An important group of enzymes is the proteases, enzymes that cleave proteins. Proteases are used, for example, in laundry detergents as stain removers, in brewing and alcohol production to increase the availability of soluble nitrogen and thus reduce fermentation time, in baking to soften gluten, and in cheesemaking to curdle milk and increase the solubility of certain proteins.

In general, enzymes are active over a narrow range of environmental conditions (temperature, pH, etc.) and are highly specific for particular substrates. This narrow range of activity limits the applicability of any particular enzyme and creates a need for a selection of enzymes having similar activities but active under different conditions or having different substrates. For example, an enzyme capable of catalyzing a reaction at 50°C may be so inefficient at 35°C that its use at the lower temperature will not be feasible. For this reason, laundry detergents generally contain a selection of proteolytic enzymes, allowing the detergent to be used over a broad range of wash temperatures and pH.

Baker's yeast (Saccharomvces cerevisiae) secretes a protein, referred to in the literature as "barrier activity" or "barrier", that acts as an antagonist of the mating pheromone α-factor. Analysis of the S. cerevisiae BAR1 gene, which encodes barrier, suggests that the protein is an aspartyl protease (MacKay et al., Proc. Natl. Acad. Sci. USA 85: 55-59, 1988). Barrier activity has been found to be heat-stable (Manney, J. Bacteriol. 155: 291-301, 1983). Barrier has not heretofore been provided in sufficient quantity or purity to be used in industrial applications.

In view of the specificity of proteolytic enzymes and the growing use of enzymes in industry, there is an ongoing need in the art for new industrial enzymes. The present invention addresses this need by providing highly enriched preparations of barrier protease and methods for producing barrier protease in useful amounts. This protease is highly heat resistant and active under acidic conditions, making it suitable for use in industrial processes that must be carried out in conditions of high temperature or low pH.

Summary of the Invention

In one aspect, the present invention provides a composition of matter comprising barrier protease having a specific activity of at least 500,000 units/mg. In another aspect, the invention provides a composition of matter comprising barrier protease having a specific activity of at least 1,000,000 units/mg. In one embodiment, the barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to isoleucine, amino acid 229. In another embodiment, the barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to asparagine, amino acid 396. In other embodiments, the barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to threonine, amino acid 422 or from leucine, amino acid 25, to proline, amino acid 425. In a fourth embodiment, the barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to tyrosine, amino acid 587.

In a related aspect, the present invention provides a method of producing barrier protease. the method comprises (a) growing in an appropriate culture medium glycosylation-defective cells of yeast transformed to express a DNA sequence encoding barrier protease and secrete barrier into the medium; (b) isolating the culture medium from the cells; (c) concentrating the isolated culture medium; (d) precipitating barrier from the concentrated medium; (e) recovering and resuspending the precipitated barrier; and (f) fractionating said resuspended precipitate to produce an enriched fraction comprising barrier protease. Within one embodiment, the yeast is Saccharomvces cerevisiae, such as a mnn1 mnn9 mutant strain. In another embodiment, the fractionating step comprises anion exchange chromatography of the resuspended precipitate. In yet another embodiment, the DNA sequence further encodes the carboxyl-terminal domain of barrier.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. Brief Description of the Drawings

Figure 1 illustrates the nucleotide sequence of a cloned S . cerevisiae BAR1 gene and the deduced amino acid sequence of the encoded barrier protein.

Figure 2 illustrates test plates for assaying barrier activity (A and B) and a control (-barrier) plate (C).

Figure 3 illustrates the subcloning of the S . cerevisiae TPI1 promoter.

Figure 4 illustrates the construction of the plasmid pSW207.

Figure 5 illustrates the construction of a vector useful for expressing barrier in transformed yeast cells.

Figure 6 illustrates an immunoblot of active fractions of barrier protease after gel filtration. Polypeptide was detected with a rabbit polyclonal antiserum raised against full-length barrier produced in. E. coli. Activity was determined with a biological assay as described herein; units are arbitrary.

Description σf the Specific Embodiments

As noted above, barrier activity acts as an antagonist to α-factor, apparently by proteolytically degrading the pheromone. Amino acid sequence analysis of the cleavage products of α-factor after exposure to barrier indicates that the enzyme cleaves at leucinelysine bonds. The action of barrier on α-factor may be represented as follows:

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Mer-Tyr

Barrier

Trp-His-Trp-Leu-Gln-Leu + Lys-Pro-Gly-Gln-Pro-Met-Tyr Barrier was also found to cleave leucine- arginine bonds.

A representative barrier amino acid sequence, shown in Figure 1, was deduced from the nucleotide sequence of a cloned BAR1 gene. Those skilled in the art will recognize that slightly different, yet functionally equivalent amino acid sequences may exist due to genetic polymorphism, or may be constructed by genetic engineering. For example, a cloned BAR1 gene or cDNA may be mutagenized to produce amino acid substitutions, deletions, or additions. These changes will generally be of a minor, conservative nature so as not to interfere with the proteolytic activity of the protein. The model of Dayhoff et al. (in Atlas of Protein Sequence and Structure 1978, Nat'1. Biomed. Res. Found., Washington, D.C.), incorporated herein by reference, may be used as a guide in selecting candidate amino acid substitutions. Such functionally equivalent forms of barrier are considered to be within the scope of the present invention.

While not wishing to be bound by theory, an analysis of the BAR1 gene (MacKay et al., 1988, ibid.) suggests that the primary translation product of 587 amino acids is cleaved after amino acid residue 24 (amino acid numbers used herein refer to the sequence shown in Figure

1) to yield a 563 amino acid mature protein with a calculated isoelectric point of 4.6. The native protein is believed to be heavily glycosylated, with a molecular weight in excess of 200 kD on SDS-polyacrylamide gels. Barrier contains nine potential N-linked carbohydrate attachment sites (sequence Asn-X-Ser/Thr), at least eight of which are believed to be glycosylated in the wild-type prorein. In addition, the carboxyl-terminal region of barrier may contain O-linked glycosylation. The predicted molecular weight of the polypeptide (exclusive of carbohydrate and assuming cleavage of the putative signal peptide) is 61.6 kD. Analysis of the BAR1 gene also suggests that the protein is organized into three functional domains. MacKay et al. (1988, ibid.) have shown that the first 372 amino acids of the putative mature protein contain regions of homology with aspartyl proteases arranged in two domains with active site aspartic acid residues at positions 63 and 287 of the primary translation product (Figure 1). The third (carboxyl-terminal) domain acts as a secretion signal to transport the protein across the cell wall and into the culture media (Welch et al., U.S. Patent Application Serial No. 07/270,933 and European Patent Office Publication EP 310,137, which are incorporated herein by reference). Deletion of the 140- most C-terminal amino acid residues does not prevent either protein export or activity. Deletion of the 165- most C-terminal residues prevents export of the remainder of the wild-type protein, but does not block proteolytic activity.

According to the present invention, barrier protease may be produced with the complete third domain (i.e. comprising amino acids 25 through 587), lacking part of the third domain (e.g. consisting essentially of amino acids 25 through 422, amino acids 25 through 425, or amino acids 25 through 447), free of the third domain (i.e. consisting essentially of amino acids 25 through 396) or lacking the second and third domains (i.e. consisting essentially of amino acids 25 through 229).

The present invention provides S. cerevisiae barrier protein at levels of purity and specific activity not heretofore obtainable. Within the present invention, barrier is provided at a specific activity of at least 500,000 units/mg of protein. In another embodiment, barrier is provided at a specific activity of at least 1,000,000 units/mg. The present invention further provides compositions of barrier protein that are substantially pure, that is at least 50% of the protein in the composition is barrier. In a preferred embodiment, barrier is provided at a purity of at least 90% as determined by analysis of polyacrylamide gel electrophoresis, Western blot and activity data. Compositions of barrier having such purity and specific activity are suitable within a variety of industrial processes requiring proteolysis at acid pH. These compositions are particularly useful for cleaving internal leucine-lysine and leucine-arginine bonds in proteins at a pH between about 2.6 and 6.8. Barrier may also be used in the production and isolation of proteins made by genetic engineering methods, for example to cleave fusion proteins at leu-lys or leu-arg bonds.

Within the present invention, barrier is obtained at high levels by producing it in host cells transformed or transfected to express a cloned DNA sequence encoding the protease. Suitable host cells include eukaryotic cells that can be grown in culture and manipulated to express cloned DNA sequences. Although cells of the yeast Saccharomyces cerevisiae are particularly preferred because they naturally produce barrier, the protease can also be made in other fungal cells (e.g. Schizosaccharomyces pombe or Aspergillus) or cells derived from multicellular organisms (e.g. cultured mammalian cell lines).

The S. cerevisiae BAR1 gene was cloned as disclosed by MacKay et al. (U.S. Patent No. 4,613,572, incorporated by reference herein in its entirety). Within the present invention, portions of the BAR1 gene encoding the mature barrier protease or proteolytically active portions thereof are subcloned and inserted into expression vectors by conventional methods.

As noted above, within the present invention it is preferred to produce barrier by culturing cells of the yeast S. cerevisiae that have been transformed to express a cloned barrier DNA sequence. When using S. cerevisiae host cells, it is advantageous to use a mating type a cell, because a cells have an endogenous BAR1 gene and do not produce α-factor, which could interfere with barrier activity or stability. It is also advantageous to use a host cell that is glycosylation-defective, that is a cell that does not add wild-type yeast carbohydrate side chains to proteins. Genes that encode functions of the S. cerevisiae glycosylation pathway include the MNN genes. A particularly preferred group of glycosylation-defective cells are those carrying the mnn1 and mnn9 mutations. mnn1 mnn9 double mutants produce glycoproteins with short, homogeneous N-linked carbohydrate side chains, whereas wild-type yeast cells produce extended, heterogeneous N- linked carbohydrate chains. Homogeneity of carbohydrate facilitates the purification of barrier. It is also preferred to use a host strain deficient in vacuolar proteases, for example a pep4 mutant.

Expression vectors for use within the present invention comprise a DNA sequence coding for barrier operably linked to a secretory signal sequence and a transcriptional promoter. The secretory signal sequence will generally be one that is syngeneic with the host cell, although this is not required. For use in yeast, the secretory signal sequence is one that will direct the barrier protease through the host cell wall and into the culture medium, making it unnecessary to disrupt the host cells to isolate the protein. Suitable secretory signal sequences in this regard include the barrier signal peptide (amino acids 1-24) and C-terminal third domain (Welch et al., ibid.), and the alpha factor pre-pro sequence (Kurjan et al., U.S. Patent No. 4,546,082; Murray et al., U.S. Patent No. 4,801,542) with or without the barrier third domain. Preferred promoters for use in yeast host cells include the promoters for the S. cerevisiae triose phosphate isomerase (TPI1) gene and the alcohol dehydrogenase I (ADHD gene or variants of these promoters. Expression vectors may also contain one or more origins of replication and selectable markers. Construction of expression vectors for a variety of host cell types is within the level of ordinary skill in the art.

As noted above, expression vectors for use within the present invention will contain the entire BAR1 gene or a functional portion thereof. In general, the vectors include at least the coding sequence for the first domain of barrier (amino acids 25-229) operatively linked to a secretory signal sequence. A preferred such construction comprises the BAR1 leader sequence or α- factor pre-pro sequence, a sequence encoding at least the first domain of barrier and a sequence encoding the third domain of barrier. Such constructions may further encode the second domain of barrier or may include tandem copies of the first domain sequence.

For barrier production, cells transformed or transfected with an expression vector as described above are grown in an appropriate culture medium under conditions suitable for expression of the barrier-encoding DNA sequence. Composition of the culture medium will be determined on the basis of the cell to be cultured, but will generally include carbon and nitrogen sources, vitamins and minerals. A selective agent will also be included if necessary for plasmid maintenance. Growth factors may also be provided. If an inducible promoter is used in the expression vector, it may be necessary to alter the media composition at a suitable point in cell growth to turn on barrier expression. Selection of media appropriate for fungal and higher eukaryotic cells is within the level of ordinary skill in the art.

For isolation of barrier from the cell culture medium, the host cells are first removed, such as by centrifugation or filtration. It is then advantageous to concentrate the medium and remove low molecular weight components by filtration using, for example, a 10 kD molecular weight cutoff membrane.

Barrier is then precipitated from the concentrated medium. A preferred method of precipitation is ethanol precipitation. Typically, barrier is precipitated by adding to the concentrated medium approximately two volumes of cold 95% EtOH and holding the mixture below 0ºC for at least 30 minutes. The resulting precipitate is recovered, for example by centrifugation.

The precipitated protein is then resuspended and fractionated, for example by anion exchange chromatography, to produce substantially pure barrier protease. A variety of anion exchange media may be used, such as derivatized dextrans, agarose, cellulose, polyacrylamide, speciality silicas, etc. The solution to be fractionated is loaded onto an anion exchange column in a weak ionic strength, neutral to slightly basic buffer and eluted with a salt gradient. In a preferred embodiment, the precipitated protein is resuspended in distilled water, combined with an equal volume of 100 mM Tris pH 8.3, and applied to a column of DEAE Fast-flow Sephadex ™ (Pharmacia, Piscataway, NJ). Protein bound to the column is eluted with a salt gradient, such as a 0-1 M NaC1 gradient. Fractions are assayed for barrier activity, and peak fractions are pooled. The pooled peak is then diluted with a slightly basic, low ionic strength buffer (e.g. 100 mM Tris pH 8.3) and fractionated by anion exchange chromatography on a Pharmacia FPLC™ system equipped with a Mono-Q™ (Pharmacia) column. Fractions are assayed for barrier activity, and the peak fractions are pooled. The resulting enriched fraction contains substantially pure barrier protease.

Other chromatographic methods, such as gel filtration, immunoaffinity chromatography or affinity chromatography on alumina, may be used in place of or in addition to anion exchange chromatography.

The purified barrier is then stored in a suitable storage buffer, such as a low ionic strength, neutral pH buffer containing 10%-50% glycerol or other stabilizing agents. The storage buffer may further contain albumin. In general, it is preferred to carry out the above-described purification procedure in the presence of a protease inhibitor so as to minimize breakdown of the barrier protein.

Barrier activity is detected in test samples using a biological activity assay based on that of Manney (ibid.). The assay can detect as little as approximately 1 ng of the enzyme. Briefly, a nutrient agar plate is overlaid with an agar solution containing the test sample, diluted as necessary, and stationary phase MATa S. cerevisiae cells. It is preferred to use cells carrying a bar1 mutation. Wells are cut in the agar, and serial dilutions of α-factor are applied to the wells. After about 24-48 hours of incubation, the plates are examined. Barrier present in test samples will reduce growth inhibition (seen as clear halos around the wells). Barrier activity is calculated as: X 1000 = units/ml

By way of example, Figure 2 shows barrier assay plates containing 1:250 dilutions of test sample (A and B) or no test sample (C). Counter-clockwise from top, the wells contain a 1 mg/ml solution of α-factor diluted at 1:75, 1:150, 1:300, 1:600, 1:1200, or distilled H₂O. In (A), the highest dilution of α-factor producing a halo is 1:150. The activity is thus:

250 x 1000 = 1,667 units/ml.

150

In (B), a halo is visible at a 1:600 dilution of α-factor. The activity is:

250 x 1000 = 417 units/ml.

600 Barrier specific activity (units of activity per mg of protein) is then calculated as activity (in units/ml) divided by protein content of the sample (in mg/ml). Protein content is determined by any of a number of conventional assay techniques.

Proteolytic activity of barrier preparations is also assayed by combining test samples with α-factor or another substrate and incubating the mixture at about 30- 37° for about 15 minutes. A typical 100 μl reaction mixture contains 50 mM sodium citrate, pH 5.0, 5 μg α- factor (ca. 30 μM) and ≤ 50 ng purified enzyme. The reaction products are separated by HPLC. Purified substrate subjected to HPLC is used as a control. The presence of α-factor breakdown products in a reaction mixture is indicative of barrier activity.

Analysis of purified barrier prepared according to the present invention demonstrated that the protein has a pH optimum of 5.0-5.3, although it exhibits detectable activity over a range of pH 1.1 to 7.9, and retains >50% activity between about pH 2.6 and 6.8. Proteolytic activity was no6 inhibited by 10 mM EDTA, 100 mM ε-amino caproic acid, 25 mM phenyl methyl sulfonyl fluoride, 500 u/μl aprotinin, 10 mM tosyl arginyl methyl ester, 10 mM tosyl lysyl methyl ester or 1 mM pepstatin A. The enzyme does not require divalent cations or other co-factors.

The following examples are offered by way of illustration, not by way of limitation.

EXAMPLES

Example 1: BARl Gene Cloning and Expression Vector

Construction

The S. cerevisiae BARl gene was cloned as disclosed by MacKay et al. (U.S. Parent No. 4,613,572, incorporated herein by reference). Briefly, a pool of plasmids containing a random mixture of yeast genomic DNA fragments derived from S. cerevisiae was constructed (Nasmyth and Tatchell, Cell 19: 753-764, 1930) using the shuttle vector YEp13 (Broach et al., Gene 8: 121-133, 1979). The resulting plasmid pool was used to transform S. cerevisiae strain XP635-10C (MATa leu2-3 leu2-112 bar1- 1 ga12; ATCC #20679) and transformants were selected for leucine prototrophy and growth on a concentration of α- factor that is inhibitory to the MATa bar1 cells. Resultant colonies were then screened for the ability to secrete barrier activity. Two colonies were found which carried both leucine prototrophy and the ability to secrete barrier. Subcloning and screening for barrier secretion localized the functional BAR1 gene sequence to a region of approximately 2.75 kb. This fragment comprises the coding sequence, nontranslated transcribed sequences, promoter, regulatory regions, transcription terminator, and flanking chromosomal sequences.

Referring to Figure 3, plasmid pM220 was used as the source of the TPI1 promoter (Alber and Kawasaki, J. Mol. APOI. Gen. 1: 419-734, 1982). E. coli RR1. transformed with pM220 has been deposited with the ATCC under accession number 39853. Plasmid pDR1107, comprising the TPI1 promoter and terminator, was constructed by first subcloning the 900 bp Bgl II-Eco RI TPI1 promoter fragment of pM220 into pIC7 (Marsh et al., Gene 32: 431-485, 1984) to generate plasmid pDRHOl. Plasmid pDR1101 was then digested with Hindd III and Sph I to isolate the 700 bp partial TPI1 promoter fragment. Plasmid pDR1100, comprising the 800 bp Xba I-Bam HI TPI1 terminator fragment of pM220 subcloned into pUC18, was cut with Hind III and Sph I. The 700 bp partial TPI1 promoter was ligated into linearized pDRHOO to produce pDR1107.

The TPI1 promoter from pM220, modified to insert an Xba I site at the 3' end of the promoter sequence, was used to replace the TPI1 promoter present in pDR1107. Plasmid pM220 was digested with Eco RI, and the 0.9 kb fragment comprising the TPI1 promoter was isolated by agarose gel electrophoresis and the ends were blunted with DNA polymerase I (Klenow fragment). Kinased Xba I linkers were added to the fragment, which was then digested with Bgl II and Xba I. This modified TPI1 promoter fragment was then ligated into the 3.4 kb Bgl II-Xba I vector fragment of pDR1107 to produce pZVllδ.

The Eco RI site that was regenerated at the 3' end of the TPI1 promoter in pZV118 was then destroyed. The plasmid was digested with Hind III and Eco RI, and the 0.9 kb fragment was isolated and ligated to a synthetic linker constructed by annealing oligonucleotides ZC708 (5'AAT TGC TCG AGT 3') and ZC709 (3' CGA GCT CAG ATC 5'). (Oligonucleotides were synthesized on an Applied Biosystems model 380A DNA synthesizer and purified by polyacrylamide gel electrophoresis). ZC708 and ZC709 were kinased and annealed essentially as described by Maniatis et al. (Molecular Cloning, A Laboratory Method, p. 122, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; incorporated herein by reference). The adapter addition eliminated the Eco RI site at the 3' terminus of the TPI1 promoter fragment and added Xho I and Xba I sites. This fragment was then joined to Hind III+Xba I- cut pUC13. The resultant plasmid was designated pZV134 (Figure 3).

The TPI1 promoter contained in pZV134 was placed into the yeast vector pDPOT. This vector was derived from the vector pCPOT (available from ATCC as an E. coli strain HB101 transformant, Accession No. 39685). As shown in Figure 4, the vector pCPOT was altered by replacing the 750 bp Sph I-Bam HI fragment containing 2 micron and pBR322 sequences with a 186 bp Sph I-Bam HI fragment derived from the pBR322 tetracycline resistance gene, to construct plasmid pDPOT. Plasmid pDPOT was modified to destroy the Sph I site and place a Not I site 5' to the Bam HI site. Oligonucleotides ZC994 (5' GAT CCG CGG CCG CAC ATG 3') and ZC995 (5' TGC GGC CGC G 3') were kinased and annealed to form an adapter with a 5' Sph I-compatible end which destroys the Sph I site, a Not I site, and a 3' Bam HI adhesive end. Plasmid pDPOT was linearized by digestion with Sph I and Bam HI. The linearized pDPOT was ligated with the ZC994/ZC995 adapter to form the plasmid PSW197 (Figure 4).

The TPI1 promoter was inserted into plasmid pSW197 to construct pSW207. Plasmid pZV134 was digested with Bgl II and Eco RI to isolate the 0.9 kb promoter fragment. The TPI1 promoter fragment and the ZC994/ZC995 adapter, described above, were ligated in a three-part ligation with pUC18 that had been linearized by digestion with Sph I and Eco RI. The resultant plasmid, pSW198, was digested with Not I and Bam HI to isolate the 0.9 kb TPI1 promoter fragment. This fragment was ligated with pSW197 which had been linearized by digestion with Not I and Bam HI. The resultant plasmid was designated pSW207 (Figure 4).

A plasmid comprising the TPI1 promoter-MFα1-BAR1 fusion was constructed as shown in Figure 5. Plasmid pZV9 (MacKay et al., U.S. Patent No. 4,613,572; available from American Type Culture Collection, Rockville, MD as a transformant in E. coli strain RR1 under Accession No.

53283) was digested with Xba I and Sal I to isolate the 767 bp BAR1 fragment. The 767 bp fragment was subcloned into Xba I+Sal I-linearized pUC13 to construct plasmid pZV16. Plasmid pZV16 was digested with Eco RI and Sal I to isolate the 651 bp BAR1 fragment. The 651 bp BAR1 fragment was ligated with a kinased Hind III-Eco RI BAR1- specific adapter (produced by annealing oligonucleotides ZC566: 5' AGC TTT AAC AAA CGA TGG CAC TGG TCA CTT AG 3' and ZC567: 5' AAT TCT AAG TGA CCA GTG CCA TCG TTT GTT AA 3') into pUC13 cut with Hind III and Sal I. The resultant plasmid, pZV96, was digested with Hind III and Sal I to isolate the 634 bp BAR1 fragment. Plasmid pM220 was digested with Bgl II and Hind III to isolate the 1.2 kb TPI1 promoter-MFα1 prepro fragment. The 3' portion of the BARl coding region was obtained by cutting pZV9 with Sal I and Bam HI to isolate the 1.3 kb BAR1 fragment. The 1.2 kb Bgl II-Hind III TPI1 promoter-MFα1 prepro fragment, the 684 bp Hind Ill-Sal I BAR1 fragment and the 1.3 kb Sal I- Bam HI BAR1 fragment were joined with Bam Hi-linearized YEp13 in a four-part ligation. A plasmid having the TPI1 promoter-MFα1-BAR1 insert in the same orientation as the Tet^R gene m the vector was designated pZV100 (Figure 5).

The TPI1 promoter-MFα1-BAR1 insert from pZV100 was subcloned into a yeast expression vector in the anti- Tet^R orientation. YEp13 was linearized by digestion with Sph I and Bam HI. The linearized vector was recircularized by ligation with a kinased ZC994/ZC995 oligonucleotide adapter having an Sph I adhesive end that destroys the Sph I site, a Not I site and a Bam HI adhesive end. The resulting plasmid, pZVl99, was linearized by digestion with Not I and Bam HI. Plasmid pZV100 was digested with Sph I and Bam HI to isolate the approximately 2.5 kb fragment containing the partial TPI1 promoter-MFα1-BAR1 fragment. The 5' portion of the TPI1 promoter was obtained as a Not I-Sph I fragment derived from pSW207. The Not I-Sph I partial TPI1 promoter fragment, the Sph I-Bam HI partial TPI1 promoter-MFα1-BAR1 fragment and the Not I-Bam HI-linearized pZV199 vector were joined in a three-part ligation. The resulting plasmid was designated pZV200. Example 2: Expression and Purification of Barrier

Protease

S. cerevisiae strain XCY93-1D (MATα Amnnl::URA3 Δmnn9::URA3 ade2 adeX leu2-3.112 Δpep4::TPlp-CAT suc2-Δ9) was transformed with pZV200 by standard procedures. Transformants were cultured in -leuDS medium (Table 1).

0.5 ml of an overnight culture was inoculated into 10 ml of fermentation medium (Table 1) adjusted to 2% glucose with glucose/trace element solution (Table 1) in a 50 ml flask. The culture was grown at 30ºC with shaking. After 24 hours, glucose was added to 2%. Samples were taken at 24, 36 and 48 hours and assayed for Barrier activity. TABLE 1

-leuDS

20 g glucose

6.7 g Yeast Nitrogen Base without amino acids (DIFCO Laboratories Detroit, MI)

0.6 g -LeuThrTrp Amino Acid Mixture

182.2 g sorbitol

After autoclaving add: 150 mg L-threonine

40 mg L-tryptophan

-LeuThrTrp Amino Acid Mixture

4 g adenine

3 g L-arginine

5 g L-aspartic acid

2 g L-histidine free base

6 g L-isoleucine

4 g L-lysine-mono hydrochloride

2 g L-methionine

6 g L-phenylalanine

5 g L-serine

5 g L-tyrosine

4 g uracil

6 g L-valine

Fermentation Medium

0.75% yeast extract

1.4% (NH₄)₂SO₄

0.27% KH₂PO₄

0.2% MgSO₄·7H₂O

0.75M Sorbitol

0.5 ml/1 vitamin solution

Vitamin Solution

0.05 mg/ml biotin

0.5 mg/ml thiamine Glucose Trace Element Solution

50% glucose containing per liter:

215.5 mg MnSO₄·1H₂O

283.6 mg FeSO₄·7H₂O

28.1 mg CuSO₄·5H₂O

and 1 g citric acid per kg glucose To assay for barrier activity, 11 ml of YEPD agar medium in a 60 x 15 mm dish was overlaid with 1.1 ml of a solution containing 0.9 ml of 0.75% agar in H₂O (melted and held at 52°C), 0.2 ml of the sample to be tested (diluted as necessary in distilled H₂O) and 1 μl of stationary phase S. cerevisiae RC629 (MATa barl-2 ade2 his6 met1 ura1 can1 cyh2 qa12) cells in YEPD. The agar was allowed to harden, and six wells (6 mm) were made in the agar. Serial dilutions of alpha factor (Sigma Chemical Co., St. Louis, MO) (13.3, 6.65, 3.325, 1.66, 8.83 and 0 ng/μl; 75 μl/well) were added to the wells, and the plates were incubated at 30°C for 24-48 hours until well-defined halos were present. The XCY93-1D[pZV200] transformant strain was found to produce 2,667 units/ml barrier activity.

For large-scale production of barrier, XCY93- lD[pZV200] cells were cultured in a 60 liter fermentor in fermentation medium at 30°C with a glucose/trace elements/citric acid feed. pH was maintained at approximately 4.0 with a 2 M NH₄OH feed. (The cells grew poorly due to a lack of adenine in the culture medium.). The cells were harvested at 38.5 hours by filtration through a 0.2 μ hollow fiber filter. The resulting cleared culture medium was concentrated 67-fold on a 10 kD molecular weight cutoff membrane filter (SIOY10 spiral cartridge; Amicon, Danvers, MA). Barrier protease secreted by the mnn1 mnn9 mutant cells was found to have a molecular weight of 92-95 kD as determined by Western blot analysis.

Proteins were precipitated from the concentrated medium by the addition of 2 volumes of cold 95% EtOH. The mixture was held at 4ºC for 30 minutes, then at -20°C for 2 hours. The mixture was then centrifuged at approximately 13,000 x g for 25 minutes to pellet the proteins. Protein pellets were resuspended in 50 ml of 63.3% EtOH, pooled, centrifuged and stored at -80°C.

The pooled protein pellet was resuspended in distilled H₂O to a final volume of 678 ml. The resulting solution was aliquoted into five 67.8 ml fractions and one 339 ml sample. These aliquots were stored at -20°C.

One aliquot (67.8 ml) was centrifuged at 27,500 x g for 20 minutes. The resulting pellet was discarded, and the supernatant was diluted with an equal volume of 100 mM Tris pH 8.3 to a final volume of 116 ml. Protein content of the solution was estimated to be 2.2 mg/ml by measurement of A₂₈₀, assuming a reading of 1.0 to indicate a protein concentration of 1 mg/ml.

The sample was loaded onto a 5 cm x 5 cm column of DEAE Fast-Flow Sephadex (Pharmacia) at a flow rate of 2 ml/minute. Bound protein was eluted from the column using a linear gradient of 0-1 M NaCl in 100 mM Tris pH 8.3 over approximately 75 minutes at a flow rate of 2 ml/minute. Two-minute fractions were collected and assayed for barrier activity. Peak fractions were pooled.

The pooled DEAE fractions were diluted 3-fold with 100 mM Tris pH 8.3 and further separated by anion exchange chromatography using an FPLC™ system equipped with a Mono-Q™ HR 10/10 column (Pharmacia). Proteins were separated using a linear gradient of 0-1 M NaCl in 100 mM Tris pH 7.6 at a flow rate of 4 ml/minute. 0.5 minute fractions were collected and assayed for barrier activity. Peak fractions were pooled. Throughout purification, samples were taken for determination of total protein content (using BCA™ protein assay reagent. Pierce Chemical Co.) and barrier activity (by bioactivity and HPLC assays). Data are presented in Table 2. Following the above-described purification, barrier specific activity was enriched over 2000-fold over yeast culture supernatant.

ND = not determined

A variety of natural and synthetic substrates were assayed for cleavage by barrier. Cleavage sites were determined by sequencing of cleavage products isolated by

HPLC. Results are shown in Table 3 (underlining indicates the location of the scissile bond). Table 3

Substrate Cleavage

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr + His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr + Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr + Trp-Leu-Gln-Leu-Lvs-Pro-Gly-Gln-Pro-Met + Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr - Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr - His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro - Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln - His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln - Trp-Leu-Gln-Leu-Lys-Pro-Gly - Trp-Leu-Gln-Leu-Lys-Pro- - Trp-His-Trp-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr + Pro-Thr-Val-Gln-Leu-Arg-Pro-Val-Gln-Val-Arg-Lys - Lys-Pro-Ile-Glu-Phe-Nphe-Arg-Leu - Ala-Pro-Ala-Lys-Phe-Nphe-Arg-Leu - Arg-Phe-Leu-Glu-Nphe-Lys-Pro-Gly-Gln-Pro - Lys-Pro-Glu-Ile-Nphe-Lys-Ser-Glu-Lys-Ile - S. cerevisiae strain XCY93-1D has been deposited with the American Type Culture Collection, Rockville, MD under the terms of the Budapest Treaty and has been assigned accession number 20996.

Additional purification was achieved by gel filtration on Sephacryl S-200 (Pharmacia). The barrier peak from the Mono-Q column was loaded onto a 200 ml bed volume S-200 column. Protein was eluted at a flow rate of 0.3 ml/minute with 50 mM Tris, pH 7.6, 0.2 M NaCl. Ten- minute fractions were collected.

Assay of the fractions from the S-200 column identified several overlapping protein peaks that exhibited barrier activity. Western blot analysis of these fractions using rabbit polyclonal antisera raised against barrier polypeptide made in E. coli demonstrated the existence of multiple, fairly discreet fragments ranging in size from about 92 kD down to about 22 kD. Barrier activity was detected over the entire size range (Figure 6).

The ca. 22 kD fragment detected in the S-200 eluant corresponds in size to a fragment that would result if barrier were able to cleave itself at the unique Leu- Lys site near the end of the first domain (amino acids 199-200 of the primary translation product) . Western blot analysis using an antibody specific for the third domain of barrier indicated that the smaller cleavage products lack at least part of the third domain. Together these results suggest that barrier can cleave itself to release a monodomain fragment that can form an active homodimer as has been reported for the HIV protease (Darke et al., J. Biol. Chem. 264: 2307, 1989; Meek et al., Proc. Natl . Acad. Sci. USA 86: 1841, 1989) and for pepsin (Bianchi et al., Biochem. Biophys. Res. Comm. 167: 339, 1990).

Example 3: Monodomain Barrier Protease

To further investigate the hypothesis that the first domain of barrier alone is sufficient for proteolytic activity, the active site aspartic acid codon at position 287 of the BARl coding sequence was mutagenized to an alanine codon using the synthetic oligonucleotide ZC437 (5' CCA GTT TTA TTA GAA TCA GGA ACC T 3' ) . Plasmid pZV9 was digested with Sal I and Bam HI to isolate the 1.3 kb fragment comprising the BAR1 coding sequence. The Sal I-Bam HI fragment was subcloned into Sal I + Bam Hi-linearized M13mp18. Single-stranded DNA from the resultant construct was mutagenized with ZC437 essentially as described by Zoller and Smith (DNA 3: 479- 488, 1984). The mutant sequence was confirmed by sequence analysis and the insert was removed by digestion with Sal I and Bam HI. Plasmid pZV9 was digested with Hind III and Sal I to isolate the ca. 1.9 kb fragment comprising the 5' non-coding region and 5' BARl coding sequence. The ca. 1.9 kb Hind Ill-Sal I fragment and the 1.3 kb Sal I-Bam HI mutant BAR1 sequence were joined with Hind III + Bam HI- linearized YEp13. The mutant BAR1 sequence in the resultant plasmid, pZV92, was confirmed by sequence analysis of the Sal I-Bam HI fragment, which was subcloned into Sal + Bam HI-linearized M13mpl8.

The Sal I-Bam HI fragment comprising the active site mutation in the BAR1 coding sequence was used to replace the coding sequence in pZV200. Plasmid pZV92 was digested with Sal I and Bam HI to isolate the 1.3 kb fragment. Plasmid pZV200 was digested with Not I and Bam HI and with Not I and Sal I to isolate the YE.pl3 vector containing fragment and the approximately 2 kb fragment comprising the TPI1 promoter, the MFα1 prepro and the 5' BAR1 coding sequence. The resulting plasmid was designated pZY109.

Plasmid pZY109 was transformed into

Saccharomyces cerevisiae strain XCY88-4D (MATa ade2 adex leu2 Δmnn9::URA3 Δmnnl::URA3 Apep4 bar1 Δsuc2). The transformants were selected on -leuDS. Barrier was purified essentially as described in Example 2.

The activation of the pZY109 mutant barrier by wild-type barrier was assayed using alpha-factor as substrate. Ten microliters (approximately 1.3 μg) of mutant barrier was pre-incubated with varying amounts of wild-type barrier, from 0.5 μl (8 ng) to 10 μl (170 ng), in a total of 30 μl for fifteen minutes at 30°C. As a control, the same amounts of wild-type barrier, from 0.5 μl to 10 μl, were pre-incubated in the absence of mutant barrier in a total of 30 μl for fifteen minutes at 30°C. The reactions were removed to an ice bath, and 10 μl of each reaction was added to a solution containing 9 μl 0.5 M sodium citrate, 76 μl of distilled water and 5 μl of a 1 mg/ml solution of alpha-factor. The reactions were incubated for fifteen minutes at 30°C and stopped by the addition of 1.5 μl concentrated H₂SO₄. The reactions were stored at -20°C. Ninety microliters of each sample was injected onto a C-18 column in a Varian 5500 HPLC equipped with an auto sampler. The samples were run using the gradient shown in Table 4.

Analysis of the HPLC tracing showed that when wild-type barrier was added to the mutant barrier, the percent of product (alpha-factor breakdown product) was greatly increased over that produced with wild-type barrier alone (See Table 5), further suggesting that barrier may self-process and that the first domain is sufficient for biological activity.

Although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

Claims We claim:

1. A composition of matter comprising barrier protease having a specific activity of at least 500,000 units/mg.

2. A composition of matter comprising barrier protease having a specific activity of at least 1,000,000 units/mg.

3. A composition according to claim 1, wherein said barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to tyrosine, amino acid 587.

4. A composition according to claim 1, wherein said barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to threonine, amino acid 422.

5. A composition according to claim 1, wherein said barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to asparagine, amino acid 396.

6. A composition according to claim 1, wherein said barrier protease comprises the amino acid sequence shown in Figure 1 from leucine, amino acid 25, to isoleucine, amino acid 229.

7. Substantially pure barrier protease.

8. Barrier protease according to claim 7, wherein said protease is at least 90% pure.

9. A method of producing barrier protease, comprising:

a) growing in an appropriate culture medium glycosylation-defective cells of yeast transformed to express a DNA sequence encoding barrier protease and secrete barrier protease into the medium;

b) isolating the culture medium from said cells;

c) concentrating the isolated culture medium; d) precipitating barrier from the concentrated medium;

e) recovering and resuspending said precipitated barrier; and

f) fractionating said resuspended precipitate to produce an enriched fraction comprising barrier protease.

10. A method according to claim 9 wherein said yeast is S. cerevisiae.

11. A method according to claim 10 wherein said yeast is a mnnl mnn9 mutant strain.

12. A method according to claim 10 wherein said yeast is S. cerevisiae strain XCY93-1D.

13. A method according to claim 10 wherein said yeast is a pep4 mutant strain.

14. A method according to claim 9 wherein said fractionating step comprises anion exchange chromatography of the resuspended precipitate.

15. A method according to claim 9 wherein said DNA sequence further encodes the carboxyl-terminal domain of barrier.

16. A method according to claim 9 wherein said DNA sequence comprises a sequence encoding the first domain of barrier operatively linked to a secretory signal sequence.