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 α-, β-, and κ-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 β-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 β-casein at pH values near 2. However, degradation of β-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 α- and κ-caseins but is unable to degrade β-casein under the same experimental conditions (27
). However, under our experimental conditions β-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.