Isolation, Purification and Characterization of the Functional Protein Bovine Prochymosin as a Biological Product Expressed by a cDNA Fragment in Escherichia coli and Saccharomyces cerevisia

摘要

Prochymosin was isolated from calf stomach and purified according to standard techniques. Fermentation studies were undertaken with the E. coli insert containing strain in an attempt to detect the cloned gene product. Presumptive evidence indicated that the gene for the enzyme calf prochymosin, which had been inserted in E. coli plasmid pBR322 at the PstI site, had been expressed under fermentation conditions. The resulting product was detected on casein polyacrylamide gel electrophoresis as a band, migrating with the dye marker, that produced a clear zone in the gel after incubation at pH 2.0 overnight. Doubie immunodiffusion techniques and affinity chromatography failed to confirm the expression of an active enzyme indicating that the level of expression was low.