Overproduction of bovine ß-casein in Escherichia coli and engineering of its main chymosin cleavage site.

G. Simons, W. van den Heuvel, T. Reynen, A. Frijters, G. Rutten, C. Slangen, M. Groenen, W.M. de Vos, R.J. Siezen

Research output: Contribution to journalArticleAcademicpeer-review

14 Citations (Scopus)

Abstract

A cDNA clone containing the entire coding region for bovine β-casein 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 phagemid library. The coding segment for mature β-casein was subcloned into the T7 expression system, in which the expression of recombinant β-casein was controlled by the T7 gene 10 promoter and ribosome binding site. High level expression of Met-β-casein to ∼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 induce secretion the coding segment for mature β-casein was coupled to the ompA translations initiation signal and signal peptide coding sequence but no secretion of the fusion protein and no processing of the signal peptide from the fusion protein was observed. Instead, the Met-β-casein could be isolated in asoluble form from E.coli cells after an osmotic shock, indicative of a periplasmic location. This procedure did not lyse the cells. The protein was purified to homogeneity after a pH 4.8 isoelectric precipitation followed by reversed-phase high-performance liquid chromatography. The β-casein cDNA was altered to change the main chymosin cleavage sitein β-casein at position 192–193 in two ways, namely from Leu–Tyr to Pro–Pro and to Leu–stop. These mutations were designed to prevent generation of the bitter peptide βcasein(193–209) by chymosin cleavage. The mutant Met-β-caseins were expressed in E.coli to the same level as wild-type Met-β-casein. Purified mutant Met-β-casein(Prol92– Prol93) was no longer hydrolysed by chymosin at the 192–193 bond.
Original languageEnglish
Pages (from-to)763-770
JournalProtein Engineering
Volume6
Issue number1
DOIs
Publication statusPublished - 1993

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