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Molecular Cloning and Expression of Bovine -Casein in Escherichia coli

Department of Food Science, University of Wisconsin, Madison 53706

T. Richardson

Department of Food Science and Technology, University of California, Davis 95616

ABSTRACT

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 -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 -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 -casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine -casein.