Investigating the use of the chymosin-sensitive sequence of kappa-casein as a cleavable linker site in fusion proteins.
The chymosin-sensitive sequence of bovine k-casein A (kappa-CN A) was investigated as a cleavable linker site between the two domains of a streptavidin-chloramphenicol acetyltransferase fusion protein. Two DNA sequences were synthesized which encode the amino acids from 101 to 107 and from 97 to 113 of bovine kappa-CN A. These sequences were separately cloned in-frame to a streptavidin expression vector used for fusion protein construction. The gene for chloramphenicol acetyltransferase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensitive linker vector forming plasmids pStCL1CAT and pStCL2CAT. The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western blot analysis of chymosin-treated cell lysates showed a pH-dependent cleavage of the fusion proteins. Fusion proteins were also bioselectively immobilized onto biotinylated controlled-pore glass beads and treated with chymosin. CAT was specifically released by chymosin treatment and was identified by SDS-PAGE.
Walsh
M K
MK
Department of Food Science, North Carolina State University, Raleigh 27695-7624, USA.
Swaisgood
H E
HE
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis; D001483:Base Sequence; D002364:Caseins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D012085:Chymosin / Q000378:metabolism*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002364:Caseins; D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D015500:Chloramphenicol O-Acetyltransferase; D012085:Chymosin