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-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 beta-casein was subcloned into the T7 expression system, in which the expression of recombinant beta-casein was controlled by the T7 gene 10 promoter and ribosome binding 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 induce 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 the signal peptide from the fusion protein was observed. Instead, the Met-beta-casein could be isolated in a soluble 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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Simons
G
G
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), Ede, The Netherlands.
van den Heuvel
W
W
Reynen
T
T
Frijters
A
A
Rutten
G
G
Slangen
C J
CJ
Groenen
M
M
de Vos
W M
WM
Siezen
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002364:Caseins / Q000235:genetics*; D002417:Cattle; D012085:Chymosin / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D014764:Viral Proteins
D002364:Caseins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012085:Chymosin