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 kappa-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 t o 107 and from 97 to 113 of bovine kappa-CN A. These sequences were se parately cloned in-frame to a streptavidin expression vector used for fusion protein construction. The gene for chloramphenicol acetyltransf erase (CAT) was then cloned in-frame to a streptavidin-chymosin-sensit ive linker vector forming plasmids pStCL1CAT and pStCL2CAT, The fusion protein was expressed in Escherichia coli and SDS-PAGE and Western bl ot analysis of chymosin-treated cell lysates showed a pH-dependent cle avage 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 an d was identified by SDS-PAGE.