OVERPRODUCTION OF BOVINE BETA-CASEIN IN ESCHERICHIA-COLI AND ENGINEERING OF ITS MAIN CHYMOSIN CLEAVAGE SITE

A cDNA clone containing the entire coding region for bovine beta-casei n A3 flanked by 53 base pairs of 5' non-coding and 358 base pairs of 3 ' non-coding sequences was isolated from a bovine mammary cDNA phagemi d library. The coding segment for mature beta-casein was subcloned int o the T7 expression system, in which the expression of recombinant bet a-casein was controlled by the T7 gene 10 promoter and ribosome bindin g site. High level expression of Met-beta-casein to approximately 20% of the total soluble proteins was obtained in Escherichia coli within 2 h after induction of T7 RNA-polymerase synthesis. In an attempt to i nduce secretion the coding segment for mature beta-casein was coupled to the ompA translational initiation signal and signal peptide coding sequence but no secretion of the fusion protein and no processing of t he signal peptide from the fusion protein was observed. Instead, the M et-beta-casein could be isolated in a soluble form from E.coli cells a fter an osmotic shock, indicative of a periplasmic location. This proc edure did not lyse the cells. The protein was purified to homogeneity after a pH 4.8 isoelectric precipitation followed by reversed-phase hi gh-performance liquid chromatography. The beta-casein cDNA was altered to change the main chymosin cleavage site in beta-casein at position 192-193 in two ways, namely from Leu-Tyr to Pro-Pro and to Leu-stop. T hese mutations were designed to prevent generation of the bitter pepti de betacasein(193-209) by chymosin cleavage. The mutant Met-beta-casei ns were expressed in E.coli to the same level as wild-type Met-beta-ca sein. Purified mutant Met-beta-casein(Pro192-Pro193) was no longer hyd rolysed by chymosin at the 192 - 193 bond.