Molecular cloning and expression of bovine kappa-casein in Escherichia coli.
A cDNA library was constructed using poly(A) +RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using 32P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein.
Kang
Y C
YC
Department of Food Science, University of Wisconsin, Madison 53706.
Richardson
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000032:analysis / Q000096:biosynthesis*; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004926:Escherichia coli; D008894:Milk Proteins / Q000032:analysis / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*
D002364:Caseins; D008894:Milk Proteins; D011994:Recombinant Proteins; D004247:DNA