Enzymatic properties and processing of bovine prochymosin synthesized in Escherichia coli

Active chymosin has been produced from calf prochymosin synthesized in E. coli. A preprochymosin cDNA clone was used to construct a plasmid, pWHA43, which expresses methionyl-prochymosin. This zymogen protein is synthesized in the bacteria in a stable but insoluble form localized in cytoplasmic inclusion bodies. Partially purified and solubilized prochymosin from E. coli can be processed by the same apparent mechanism as authentic calf prochymosin upon activation with acid. The chymosin thus derived from E. coli showed the same substrate specificity and the same kinetics of activation as that of chymosin derived from purified calf stomach prochymosin. These results also demonstrate that the bovine prochymosin synthesized in E. coli can be reconstituted and activated to a form having the functional properties necessary for an industrial milk coagulant to be used in cheese manufacturing.