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Applied and Environmental Microbiology, May 2004, p. 2588-2595, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2588-2595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

Rosario Muñoz,¹ José L. García,² Alfonso V. Carrascosa,¹ and Ramon Gonzalez^1*

Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC),¹ Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain²

Received 22 October 2003/ Accepted 15 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with -, ß-, or -casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bovine pepsin is a member of the pepsin-like family of aspartic peptidases found in the fourth stomach of cows. Peptidases belonging to this family exhibit optimal activity at an acidic pH and contain two active-site aspartate residues required for function. Like other gastric proteinases, bovine pepsin is synthesized as an inactive precursor or zymogen, pepsinogen. Pepsinogen contains a prosegment at the N-terminal end, which prevents access of the substrate to the active site and maintains the enzyme in its inactive form. The conversion of pepsinogen to active pepsin is initiated by acidic conditions and involves several conformational changes and bond cleavage steps that result in proteolytic removal of the prosegment and release of the active site (20).

The main industrial application of bovine pepsin is in cheese making; this enzyme is either naturally present in calf stomachs or is added as a less expensive complement in calf chymosin preparations (2, 6). The fluctuations in the availability and price of calf rennet some years ago stimulated the search for alternative milk coagulants; this search included cloning and expression in several microorganisms of a cDNA coding for calf prochymosin (3, 17, 28). Recombinant bovine chymosin was first commercialized in 1990 (5); sales of this compound quickly accounted for more than one-half of the market for rennet, and several versions, produced from genetically engineered Escherichia coli, Kluyveromyces lactis, or Aspergillus niger, became available. The recombinant rennets have been shown to be equivalent to natural rennet (18). After the bovine spongiform encephalopathy crisis in Europe, food safety considerations also supported the use of recombinant rennet instead of natural rennet. Whereas milk safety has been clearly documented by both epidemiological and experimental data (http://www.tseandfoodsafety.org/position_papers/position_paper_on_the_safety_o/tafs_position_paper_on_the_2.pdf), the safety of the abomasum from ruminants is inferred based partially on a number of assumptions, such as the quality of feed and the procurement of the abomasum (http://www.emea.eu.int/pdfs/human/bwp/033702en.pdf). Indeed, prions have been detected (although no infectivity has been titrated) in the abomasum of experimentally infected small ruminants (http://europa.eu.int/comm/food/fs/sc/ssc/out296_en.pdf).

Its limited proteolytic activity makes chymosin the best milk-clotting enzyme in terms of curd yield, and in addition, the off-flavors often associated with excess proteolysis are absent. However, ripening of cheese requires additional proteolytic activities, which come in part from cheese microbiota, including the starter cultures used for acidification and in part from the residual proteolytic activity of the enzymes used for milk clotting. The main changes attributable to proteolysis during cheese ripening are changes in the texture and flavor. The effect on flavor is due to the release of peptides and amino acids, which, in turn, can be the substrates for further reactions resulting in aroma compounds, such as deamination, decarboxylation, or desulfuration (7). The acceptable degree of proteolysis during ripening depends on the type of cheese, and several strategies have been proposed for accelerating this process in order to reduce production costs. These strategies include the use of elevated temperatures (13), addition of proteolytic enzymes (13), and addition of bacteriocin-producing starters (8, 15, 16). It has been suggested that the different enzyme specificities of bovine pepsin and chymosin may contribute favorably to the quality of cheeses made with natural rennet preparations containing pepsin (30).

Several pepsin-encoding genes, including the genes from pigs (26) and chickens (21), have been cloned, and some of them have been expressed in microorganisms (26); however, remarkably, the gene coding for bovine pepsin has not been completely described so far. Only the N-terminal amino acid sequence of bovine pepsinogen (10) and a partial genomic sequence erroneously labeled as the sequence encoding bovine pepsinogen have been determined previously (14). In this paper we describe cloning of a complete cDNA encoding bovine pepsinogen and expression of this cDNA in bacterial and yeast cells. The use of the technology described in this work is covered by Spanish patent application 200300179.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials.
cDNA clones from two different bovine cDNA libraries were used as starting material for construction of a complete cDNA coding for bovine pepsinogen. Clones 1Abo01C03, 1Abo04B07, 1Abo09A06, 1Abo10A02, 1Abo11A02, 1Abo15A08, 1Abo15G06, and 1Abo16A03 (GenBank accession numbers BG937426, BG937636, BG937863, BG937943, BG938081, BG938289, BG938334, and BG938347, respectively) were obtained from a bovine abomasum cDNA library (1Abo) in the Uni-ZAP XR vector (Stratagene, La Jolla, Calif.) and were kindly provided by S. Moore and C. Hansen from the University of Alberta, Edmonton, Alberta, Canada. Clones MARC3BOV 85L15, MARC3BOV 103I10, MARC3BOV 103G10, and MARC3BOV 105H9 (GenBank accession numbers BF774958, BM106242, BM106232, and BM106659, respectively) were obtained from a mixed-tissue bovine cDNA library (MARC3BOV) in pCMV-SPORT6 (Invitrogen, Leek, The Netherlands) and were obtained from the Children's Hospital Oakland Research Institute. Purified milk proteins and other chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.). Stabo 230, a commercial enzyme solution containing approximately 85% bovine pepsin and 15% chymosin, was kindly provided by Christian Hansen A/S (Hoersholm, Denmark).

Microbial strains, plasmids, and growth conditions.
Escherichia coli DH5F' [F' endA1 hsdR17 (r_k^–m_k⁺) supE44 thi-1 recA1 gyrA(Nal^r) relA1 (lacIZYA-argF)U169 deoR (80dlac(lacZ)M15; Promega] was used for all DNA manipulations, as well as for pepsinogen production. E. coli JM109(DE3) [endA1 recA1 gyrA96 hsdR17 supE44 relA1 thi (lac-pro) F' (traD36 proAB⁺ lacI^q lacZM15) cI857 ind1 Sam/nin5 lacUV5-T7 gene 1; Promega] and Saccharomyces cerevisiae BY4741 (MATa his3-1 leu2-0 met15-0 ura3-0) were used for expression of recombinant bovine pepsinogen. pIN-III(lpp^p-5)A3 and pT7-7, two expression vectors carrying isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible promoters, were used for expression in E. coli strains (11, 25). pYES2, an episomal expression vector for S. cerevisiae carrying the GAL1 galactose-inducible promoter, was used for expression in yeast.

E. coli strains were cultured in Luria-Bertani (LB) medium (22) at 37°C and 200 rpm. When required, ampicillin was added to the medium at a concentration of 100 µg ml^–1. Yeast cells were cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C.

Recombinant DNA techniques.
Restriction endonucleases and T4 DNA ligase were obtained from Roche (Basel, Switzerland) and were used according to the recommendations of the supplier. Gel electrophoresis analyses of plasmids, restriction fragments, and PCR products were performed in agarose gels as described previously (22). Bacterial plasmids were purified by the alkaline sodium dodecyl sulfate (SDS) method (22). Yeast chromosomal DNA was purified by the method of Querol et al. (19). In vivo excision of phagemids from LambdaZAP clones was performed as suggested by Stratagene. Insertion of PCR fragments into preexisting constructs was performed by using a QuikChange kit from Stratagene, as described by Wang and Malcolm (29). PCR amplification was performed by using Pfu DNA polymerase (Stratagene) and the instructions of the supplier. The sequences of the primers used in this work are shown in Table 1.

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TABLE 1. Sequences of the primers used in this study for PCR amplification or for site-directed mutagenesis

DNA sequencing was carried out with an ABI Prism 377 DNA sequencer (Applied Biosystems, Inc., Branchburg, N.J.). Sequence similarity searches were carried out by using the Basic Local Alignment Search Tool (BLAST) (1) with the EMBL and GenBank databases.

Transformation of microorganisms.
Transformation of E. coli was performed by electroporation (22), and the transformants were selected on LB medium supplemented with ampicillin (100 µg ml^–1). Transformation of S. cerevisiae was performed by the method described by Gietz and Woods (9). Yeast transformants were selected on SD medium without uridine or uracil (0.67% yeast nitrogen base [Difco, Detroit, Mich.], 2% glucose, 60 mM leucine).

Induction and preparation of cell extracts.
Bovine pepsinogen cDNA was expressed in E. coli as follows. Bacterial cells harboring either the original vector or the recombinant plasmid were grown in LB broth supplemented with ampicillin (100 µg ml^–1) at 37°C and 200 rpm to an optical density at 600 nm of 0.6. The cultures were subsequently shifted to 20 or 37°C, and synthesis of bovine pepsinogen was induced by adding 0.5 mM (final concentration) IPTG. At different times, samples of the cultures were harvested by centrifugation (10,000 x g, 5 min), and the bacterial cells were recovered in 50 mM Tris-phosphate buffer (pH 7.0) and disrupted by sonication. The insoluble fractions were separated by centrifugation (15,000 x g, 15 min), and the supernatants were used for enzyme assays.

Expression in S. cerevisiae was induced with galactose as follows. Yeast cells harboring either pYES2 or pBP05 were grown on SD medium at 30°C and 150 rpm until the mid-exponential phase and then transferred to induction medium. Depending on the experiment, a culture containing 2 x 10⁷ cells per ml was transferred to SG minimal induction medium (0.67% yeast nitrogen base [Difco], 2% galactose, 60 mM leucine) or to YPG rich induction medium (1% yeast extract, 2% peptone, 2% galactose) and incubated under the same conditions. Samples were withdrawn at different times, the cells were separated by centrifugation (12,000 x g, 5 min), and the supernatants were assayed for proteolytic activity. In order to obtain the cell extracts, about 10⁸ cells were harvested by centrifugation at 10,000 x g for 5 min and resuspended in 500 µl of 50 mM Tris-phosphate buffer (pH 7). The cell suspension was vortexed at full speed for 5 min in the presence of 0.5 g of glass beads. Insoluble material was removed by centrifugation at 12,000 x g for 5 min, and the supernatants were used to measure cell-associated proteolytic activity.

Renaturation of inclusion bodies.
Extracts of E. coli JM109(DE3) cells harboring plasmid pBP06 induced overnight at 37°C with IPTG were used as the starting material for renaturation of the inclusion bodies. Purification and renaturation of the inclusion bodies were performed as described previously for recombinant bovine chymosin expressed in E. coli (4).

Milk-clotting assays.
Supernatants of galactose-induced S. cerevisiae cultures were dialyzed and freeze-dried. Pepsinogen was activated to pepsin as previously described (12). Briefly, 0.3 M HCl was added to the solution until the pH was 2.0, and after incubation for 10 min at room temperature the pH was raised to 6.0 by adding a cold solution of 4 M sodium acetate. Changes in the pH of the solution were continuously monitored with a pH meter. Milk-clotting activity was assayed in microtiter plates as described by Emtage et al. (4). Each well contained 100 µl of 12% (wt/vol) dried skim milk, 20 mM CaCl₂, 25 mM sodium phosphate buffer (pH 6.3), and an appropriate dilution of the commercial or activated recombinant enzyme. The plates were incubated at 37°C and after 30 min were inverted to allow nonclotted milk to drain. Clotting activity was confirmed by the presence of a white coagulate at the bottom of the well and was recorded by scanning the plates against a black background with a Scanjet 5470c scanner (Hewlett-Packard, Camas, Wash.).

Protease assays.
The amount of functional pepsinogen in a cell extract or in the culture medium was estimated by the method of Kasell and Meitner (12) by using bovine hemoglobin (Sigma-Aldrich) as the substrate. Briefly, 350 µl of hemoglobin substrate prepared as described by Kasell and Meitner (12) was incubated with 350 µl of enzyme diluted in 0.01 M HCl-0.1 M NaCl (pH 2.0) at 37°C for 30 or 60 min. The reaction was stopped by adding 700 µl of 5% trichloroacetic acid, the mixture was incubated for 15 min on ice and centrifuged at 15,000 x g for 15 min, and the supernatant was used to determine the optical density at 280 nm. Activities were expressed as equivalents of porcine pepsin (Sigma-Aldrich) assayed under the same conditions. The amount of active pepsin after pepsinogen activation (see above) was estimated as follows. First, 125 µl of azocasein substrate (2% azocasein in 50 mM sodium phosphate buffer [pH 6.0]) was incubated with 75 µl of enzyme diluted in 50 mM sodium phosphate buffer (pH 6.0). The reaction was stopped by incubation with 0.6 ml of 10% trichloroacetic acid for 15 min at 4°C, the reaction mixture was centrifuged at 12,000 x g for 15 min, and 0.6 ml of the supernatant was mixed with 0.7 ml of 1 M NaOH before the optical density at 440 nm was recorded. Isolated milk proteins were digested as described by Ustunol and Zeckzer (27), with minor modifications. Briefly, -, ß-, and -caseins (Sigma-Aldrich) were dissolved in 0.1 M phosphate buffer (pH 6.7) at a final concentration of 2 mg ml^–1. Each standardized protease solution was diluted 10-fold in the same buffer and allowed to react overnight at 30°C. The hydrolysis profiles were visualized by SDS—15% polyacrylamide gel electrophoresis (PAGE).

Nucleotide sequence accession number.
The sequence of the reconstituted cDNA determined in this study has been deposited in GenBank database under accession number AY330769.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of a complete version of bovine pepsinogen cDNA.
Sequences corresponding to human, camel, and pig pepsin-encoding genes, as well as to bovine chymosin-encoding genes, were used to perform a BLAST search of sequences potentially coding for bovine pepsin. In this way two cDNA libraries containing partial bovine pepsin cDNA sequences were identified in the expressed sequence tag database. Sequence analysis of the 1Abo and MARC3BOV clones showed that they contained the 3' end of the cDNA, including the poly(A) tail. Comparison of the hypothetical sequence of the cDNA clones with the sequence described previously for the first 110 amino acids of pepsinogen (10) revealed that the clones from the MARC3BOV library had a 118-bp internal deletion. However, clones from the 1Abo library did not have any internal deletion. Clones MARC3BOV 103I10 and 1Abo04B07 were used to obtain a complete version of bovine pepsinogen cDNA. Figure 1 shows an alignment of the N-terminal sequence of pepsinogen with the sequences encoded by both cDNA.

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FIG. 1. Sequence alignment showing the experimentally determined N-terminal sequence of bovine pepsinogen (BovPgn) (accession number P00792), the hypothetical translated sequences of cDNA clones 1Abo04B07 (04B07) (accession number BG937636) and MARC3BOV 103I10 (103I10) (accession number BM106242), the sequence of rabbit pepsinogen III (RabPgn) (accession number AAA85370), and the sequence previously assigned to bovine pepsinogen exons 6, 7, and 8 (P111) (accession number JT0398). The nucleotides that are different in the bovine pepsinogen exon 6, 7, and 8 and rabbit pepsinogen III sequences and the bovine pepsinogen cDNA clone sequence are underlined in the bovine pepsinogen exon 6, 7, and 8 sequence. The conserved positions for the introns in the genomic sequence for most mammalian aspartic protease genes are indicated by vertical lines. The sequence downstream of the 118-bp internal deletion in MARC3BOV 103I10 has been translated in a different frame. The sequences of both cDNA clones were experimentally determined in this work because the sequence in the GenBank database is a partial sequence.

The strategy used to construct a complete version of the bovine gene coding for pepsinogen is summarized in Fig. 2. A DNA fragment containing the sequence coding for bovine pepsinogen that is missing in clone 1Abo04B07 was generated by PCR performed with primers cDNA-PB2 and cDNA-pB3 by using MARC3BOV 103I10 as the template. Primer cDNA-PB3 contains the ATG start codon and the sequence coding for the four N-terminal amino acids of pepsinogen. This PCR fragment was then inserted into 1Abo04B07 by using a QuikChange kit from Stratagene, as described in Materials and Methods. The resulting plasmid, pBP01, contained a cDNA sequence coding for a complete version of bovine pepsinogen in the phagemid vector pBluescript SK(–).

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FIG. 2. Construction of plasmid pBP01 containing a complete version of bovine pepsinogen cDNA. The 118-bp internal deletion on MARC3BOV 103I10 is indicated by a dashed line. The positions of primers cDNA-PB2 and cDNA-PB3 are indicated by arrows. Sequences arising from the insert in MARC3BOV 103I10 are indicated by solid arrows, and sequences arising from the insert in 1Abo04B07 are indicated by open arrows. Vector sequences are represented by a single line [pBluescript SK(–)] or a double line (pCMV-SPORT6). pgnA, bovine pepsinogen cDNA sequences; E, EcoRI; H, HindIII; N, NcoI; P, PstI; S, SalI; Sc, ScaI; X, XhoI. (Sc) indicates a ScaI site absent in the MARC3BOV 103I10 clone due to the 118-bp internal deletion.

Sequence analysis.
The peptide sequence of bovine pepsinogen is 55% identical to the calf prochymosin sequence and 83 and 82% identical to the pig and human pepsinogen sequences, respectively. The levels of similarity are 94% for pig pepsinogen, 91% for human pepsinogen, and 71% for calf prochymosin. These values are very similar to those obtained with the corresponding mature peptides. Two copies of the consensus sequence for eukaryotic aspartic proteases, flanking the aspartate residues of the catalytic site as described in PROSITE entry PS00141 (24), are present in the deduced sequence of bovine pepsinogen at positions 75 to 86 and 258 to 269. Remarkably, although the N-terminal sequence of the cDNA is identical to the known sequences of pepsinogen genes, there are several differences between the previously published partial cDNA sequence of exons 6, 7, and 8 of the bovine pepsinogen gene (14) and the cDNA sequence described here (Fig. 1) (the overall genomic structures of most mammalian aspartic proteases are similar, with eight introns at conserved positions). Two years ago we used the partial pepsinogen sequence described by Lu et al. (14) to amplify bovine pepsinogen gene sequences from calf genomic DNA (unpublished data). To our surprise, the amplified DNA sequences (accession number AY442187) clearly differed from the sequences described previously. In fact, the coding region of this fragment is identical to the pepsinogen cDNA elucidated in this work (data not shown). In order to solve this puzzle, we performed further sequence comparisons of these three exon sequences with the whole nucleotide databases using BLAST. Surprisingly, this analysis revealed 100% identity between the sequence of the three exons and the sequence of the rabbit pepsin gene, suggesting that the exons actually are rabbit pepsinogen exons and not bovine pepsinogen exons as described previously (Fig. 1).

Expression of bovine pepsinogen in E. coli.
To express bovine pepsinogen in E. coli, the cDNA coding for pepsinogen was PCR amplified from pBP01 by using Pfu DNA polymerase and primers Xba-PB and Hind-PB. Primer Xba-PB is based on the sequence of plasmid pIN-III(lpp^p-5)A3 and contains the ribosome binding site domain, as well as the initial start codon and the sequence encoding the first five amino acid residues of pepsinogen, which were designed by preferred codon usage by E. coli. Primer Hind-PB is also based on the sequence of pIN-III(lpp^p-5)A3 and contains the sequence encoding the six last amino acid residues of pepsinogen and several stop codons arranged in tandem (Table 1). The purified PCR fragment was digested with XbaI and HindIII, whose target sequences were incorporated into the primers and cloned into pIN-III(lpp^p-5)A3 digested with the same restriction enzymes. The resulting plasmid, pBP03, contained bovine pepsinogen cDNA under control of the lpp^p-5 and lac^po promoters, which can be induced at high levels by IPTG (11). The sequence and the site of insertion of the cDNA were verified by restriction analysis and DNA sequencing. Cell extracts were prepared from E. coli DH5 cells harboring recombinant plasmid pBP03. To detect the recombinant protein, cell extracts were analyzed by SDS-PAGE (Fig. 3A). Control cells containing the pIN-III(lpp^p-5)A3 vector plasmid alone did not show expression over the 4-h time course analyzed, whereas expression of an additional 40-kDa protein was apparent with E. coli DH5 cells harboring pBP03. The molecular mass of this protein was in good agreement with the molecular mass deduced from the nucleotide sequence of the bovine pepsinogen cDNA (40 kDa).

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FIG. 3. SDS-PAGE analysis of soluble cell extracts of IPTG-induced cultures of E. coli JM109(DE3) bearing recombinant plasmids. (A) Expression on pIN-III(lppp-5)A3. Lane 1, E. coli DH5/pIN-III(lppp-5)A3; lane 2, E. coli DH5/pBP03. (B) Expression on pT7-7. Lane 1, E. coli JM109(DE3)/pT7-7; lane 2, E. coli JM109(DE3)/pBP06. The arrows indicate the position of the overproduced protein. The 12% polyacrylamide gels were stained with Coomassie blue. The positions of molecular mass markers (SDS-PAGE standards; Bio-Rad) are indicated on the left.

Cell extracts were also assayed for protease activity against bovine hemoglobin at low pH. Cell extracts of E. coli DH5 harboring recombinant plasmid pBP03 exhibited protease activity ranging from 0.2 µg (noninduced conditions) to 0.5 µg (induced conditions) of porcine pepsin equivalents per ml of culture, whereas no activity was detected in cell extracts prepared from control cells containing the vector plasmid.

To improve the production of recombinant bovine pepsinogen, we transferred the bovine pepsinogen cDNA to the expression vector pT7-7. By using the strategy described above, an XbaI/HindIII-digested PCR fragment containing bovine pepsinogen cDNA was cloned in pT7-7 digested with the same restriction enzymes. The resulting plasmid, pBP02, was used as a template for PCR amplification with Pfu DNA polymerase and primer PT7-PB. This primer provides the original ribosome binding site from pT7-7. The amplification product was digested with DpnI to degrade the DNA from the original plasmid, pBP02, and the amplified product was transformed into the expression strain E. coli JM109(DE3). The resulting plasmid, pBP06, contained bovine pepsinogen cDNA under control of the T7 RNA polymerase-inducible 10 promoter.

Several conditions were tested to induce bovine pepsinogen synthesis by E. coli JM109(DE3) cells harboring recombinant plasmid pBP06. The values obtained for pepsinogen activity were 2 orders of magnitude greater than the values obtained for pBP03-transformed cells under similar culture conditions (Table 2). Overnight (16-h) induction at 20°C resulted in the maximum functional pepsinogen production. A similar conclusion was drawn from an SDS-PAGE analysis. As shown in Fig. 3B, induction at 20°C for 16 h gave rise to a conspicuous 40-kDa protein band. We found that most, if not all, of the protein was recovered from the supernatant of the cell extracts (Fig. 3B, lane 2), indicating that it was in a soluble form. The 40-kDa protein was absent in the supernatants prepared from either an uninduced bacterial culture (Fig. 3B, lane 1) or E. coli cells harboring only the vector plasmid (data not shown). We observed a faint band of the same size with cell extracts from cultures induced for 4 h at 20 and 37°C (data not shown). In the case of the culture induced overnight at 37°C, we observed an apparent 40-kDa band in the total cell extract, indicating that the protein was in an insoluble form, probably inclusion bodies, and this could explain the lack of proteolytic activity. The formation of inclusion bodies was verified by direct observation of the induced cultures by phase-contrast microscopy. These bodies were observed in cultures induced overnight at both 20 and 37°C; large amounts of functional pepsinogen were recovered in cultures induced at 20°C, whereas no functional protein was detected in cultures induced at 37°C. Several unsuccessful attempts were made to recover the functional protein from the inclusion bodies, based on the procedure described for recombinant bovine chymosin (4).

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TABLE 2. Proteolytic activities in cell extracts of E. coli JM109(DE3) transformed with pBP06 under different IPTG induction conditions

Expression of bovine pepsinogen in S. cerevisiae.
To produce recombinant bovine pepsinogen in S. cerevisiae, a SacI/XhoI fragment from pBP01 containing pepsinogen cDNA was cloned into pYES2, a yeast episomic expression vector, which was digested with the same enzymes. The resulting plasmid, pBP04, contained the bovine pepsinogen cDNA under control of the yeast GAL1 gene promoter, which is induced by galactose and is repressed by glucose. When this system was used, we were unable to detect any proteolytic activity in either the culture broth or cell extracts from pBP04-containing yeast cells that were induced by galactose in SG medium.

A different approach was used to promote secretion of pepsinogen by construction of a transcriptional fusion of pepsinogen cDNA with the alpha-factor secretion signal of S. cerevisiae. To do this, the sequence encoding the alpha-factor secretion signal was PCR amplified from S. cerevisiae BY4741 genomic DNA by using primers BP05A and BP05B. The resulting PCR product was used to introduce the secretion signal into pBP04, which was fused upstream of the sequence coding for bovine pepsinogen, with a QuikChange kit from Stratagene, as described in Materials and Methods. The resulting plasmid, pBP05, and pBP04 were introduced into S. cerevisiae BY4741 by transformation. The transformants were tested for pepsinogen production and secretion under different induction conditions (Table 3). Only yeast strains harboring plasmid pBP05 secreted pepsinogen into the culture medium. As expected, the amount of pepsinogen secreted was larger under galactose induction conditions (SG or YPG medium) than under glucose repression conditions (SD or YPD medium). However, the yield was clearly increased by using complex medium (YPG medium). A very low level of pepsinogen production was detected in cell extracts, and only induced cultures containing cells carrying plasmid pBP05 had intracellular pepsinogen levels slightly greater than the background level (data not shown). There were no significant differences in intracellular pepsinogen levels between cells carrying pYES2 and cells carrying pBP04. The time course of induction on YPG medium for cells carrying pBP05 was also investigated (Fig. 4); most of the pepsinogen was released after between 6 and 24 h of induction, but the levels continue to increase until 72 h.

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TABLE 3. Proteolytic activities in the supernatants of S. cerevisiae strains transformed with different plasmids

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FIG. 4. Time course of extracellular pepsinogen formation in yeast cultures transferred to YPG induction medium. Symbols: •, pYES2-transformed cells; , pBP05-transformed cells.

Clotting activity.
The clotting activity of recombinant bovine pepsinogen produced in S. cerevisiae was determined as described by Emtage et al. (4). Twofold dilutions of either recombinant acid-activated pepsinogen or Stabo 230 were added to consecutive wells starting with an azocasein hydrolytic activity equivalent to 4 µg of commercial porcine pepsin. Figure 5 shows the results of this experiment, which indicated that the clotting activity of recombinant pepsin is similar to that of Stabo 230.

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FIG. 5. Milk-clotting activity of native pepsinogen (Stabo 230) or recombinant pepsinogen obtained from yeast strains carrying plasmid pBP05. The amount of enzyme used in each column is expressed in microgram equivalents of porcine pepsin.

Proteolytic activity against isolated milk proteins.
We investigated the activities of native pepsin and S. cerevisiae-produced recombinant pepsin with purified cow milk caseins and compared the activities under similar conditions. We studied the extent and profile of hydrolysis of purified -, ß-, and -caseins (Sigma-Aldrich) by using Stabo 230 or acid-activated recombinant pepsinogen solutions. The enzyme solutions were previously adjusted to contain similar proteolytic activities against azocasein. Figure 6 shows the peptide profiles of caseins after overnight incubation at 30°C in the presence of Stabo 230 and in the presence of recombinant pepsin secreted by S. cerevisiae. Both proteases showed the same peptide pattern for the three substrates used, and although the extent of hydrolysis with the recombinant protein was less than that with Stabo 230 (Fig. 6B), this may have been due to a small difference in the activity units used for the assay.

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FIG. 6. SDS-PAGE analysis of isolated cow milk caseins treated with Stabo 230 or recombinant bovine pepsin (15% polyacrylamide gels). (A) Proteolytic activity with -casein; (B) proteolytic activity with ß-casein; (C) proteolytic activity with -casein. Lane 1, control; lane 2, Stabo digestion; lane 3, soluble extract of S. cerevisiae carrying pBP05. The gels were stained with Coomassie blue. The positions of molecular mass markers (SDS-PAGE standards; Bio-Rad) are indicated on the left.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Based on the results described above, we concluded that the complete cDNA sequence encoding true bovine pepsinogen was cloned and determined for the first time. This conclusion was based on (i) the identity between the deduced protein sequence and the known N-terminal sequence of bovine pepsinogen A, (ii) the high level of similarity between this sequence and that of pepsinogens from other mammalian species, (iii) the size of the recombinant protein expressed in E. coli, (iv) the proteolytic and milk-clotting activities of the recombinant protein, and (v) the comparison of casein digestion profiles with the profiles produced by natural bovine pepsin. In spite of the puzzling noticeable differences between the partially described sequences of bovine pepsinogen exons 6, 7, and 8 and the cDNA sequence described in this paper, we demonstrated that the previously described sequences exhibit 100% identity with the rabbit pepsinogen gene, suggesting that they were most likely obtained from a contaminated cDNA library. It should be stressed that Lu et al. (14) failed to clone the bovine chymosin gene from their library, even when they used the homologous probe. In our case, we eliminated contamination since the cDNA fragments described here were derived from two different bovine cDNA libraries and are identical in the shared regions. Moreover, the hypothetical sequence translated from the cDNA sequence is identical to the first 110 amino acids of pepsinogen, and the coding sequence is identical to the coding sequence of the bovine genomic DNA fragment previously cloned by us (data not shown) (accession number AY442187).

Recombinant bovine pepsinogen has been successfully produced in E. coli and S. cerevisiae. In E. coli the best expression results were obtained by using strain JM109(DE3) as the host, pT7-7 as the expression vector, and overnight induction with IPTG at 20°C. Under these optimal conditions, pepsinogen production in cell extracts, expressed in porcine pepsin equivalents, was more than 40 mg/g (dry weight). The highest levels of yeast-expressed pepsinogen were obtained by using a transcriptional fusion of bovine pepsinogen cDNA with the S. cerevisiae alpha-factor secretion signal and a complex medium with galactose as the only carbon source (YPG medium) for induction. Even though the yeast expression levels were lower than those in E. coli, the use of pBP05 has the advantage that the recombinant pepsinogen is recovered from the culture broth. The secretion of the molecule facilitates both purification and production in a continuous culture system.

The lack of pepsinogen production in yeast carrying plasmid pBP04 may have been due to transcriptional problems. In this plasmid the distance between the transcription start site and the start codon is greater than the distance in pBP05; this is due to the presence of pBluescript multiple-cloning-site sequences that have not been removed. Nevertheless, we cannot exclude the possibility that secretion is necessary to avoid degradation by intracellular yeast enzymes.

All attempts to recover functional pepsinogen from E. coli inclusion bodies by using the strategies designed for recombinant bovine chymosin (4) have been unsuccessful. However, if the large amounts of protein obtained as inclusion bodies in cultures induced overnight at 37°C are taken into account, fine-tuning of solubilization and refolding protocols for recombinant bovine pepsinogen is a potential target for improving yield and purity.

One of the main potential applications of the recombinant enzyme is to accelerate ripening in cured cheese or to produce cheeses with properties similar to those made with rennet, which naturally contains both chymosin and pepsin. Indeed, it has been shown that ripening of Grana cheese is accelerated or improved by using mixtures containing 10% bovine pepsin compared to the ripening of cheese made by using recombinant chymosin alone (30). Remarkably, successful cheese production has been reported when 97% pure pepsin preparations were used (2). To learn more about this possibility, we compared the yeast-expressed recombinant bovine pepsinogen (after acid-induced activation) with a commercially available pepsin-rich preparation from bovine abomasum. The ratios of milk clotting to general proteolytic activity, as measured with hemoglobin or azocasein, were similar for the two enzymes. When purified -, ß-, and -caseins were used, the results were also similar in terms of the degree of proteolysis and the main hydrolysis products. Only in the case of ß-casein was there a small difference in the degree of hydrolysis, and this was probably due to slightly different amounts of enzyme used for the assay. Fox and Wallace (7) showed that pepsin can hydrolyze ß-casein at pH values near 2. However, degradation of ß-casein by pepsin is dependent not only on pH but also on the incubation temperature, and the protein is degraded more rapidly at 2°C than at 32°C. It has been suggested that pepsin is capable of degrading both - and -caseins but is unable to degrade ß-casein under the same experimental conditions (27). However, under our experimental conditions ß-casein was degraded by both the native bovine pepsinogen and the recombinant bovine pepsinogen, which generated the same peptide profile.

In conclusion, this is the first time that a recombinant bovine pepsinogen has been synthesized, and our findings pave the way for using this enzyme as an alternative in cheese making; use of this recombinant enzyme has the advantage that the recombinant pepsin should be free from potential pathogenic agents arising from animal tissues, particularly the causative agent of bovine spongiform encephalopathy. In addition, like recombinant bovine chymosin, recombinant pepsin would be more acceptable than the native enzyme to vegetarian consumers or to people subject to food restrictions due to religious beliefs. Finally, other potential applications of bovine pepsin include the release of bioactive peptides from casein macropeptide or caseins (23) or use as a general-purpose protease.

 
We are grateful to C. Hansen and S. Moore for kindly providing cDNA clones from the 1Abo library, to E. Cebollero for help with yeast transformation, to F. Jorganes and A. Fernandez for technical assistance, and to M. Sheehan for correcting the English version.

 
* Corresponding author. Mailing address: Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. Phone: 34-915622900, ext. 359. Fax: 34-915644853. E-mail: rgonzalez{at}ifi.csic.es.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, May 2004, p. 2588-2595, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2588-2595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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