PRODUCTION OF NATIVE CREATINE KINASE-B IN INSECT CELLS USING A BACULOVIRUS EXPRESSION VECTOR

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZl-BCK). Sf9 cells infected with this re combinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtr ation chromatography the recombinant protein behaved like authentic di meric human BB-CK protein. Studies with a newly produced monoclonal an tibody (CK-BYK/2 1E10) directed against an epitope in the N-terminus o f the protein confirmed the identity of the product. The recombinant B B-CK protein was purified to over 99% homogeneity from the total prote in extract of AcDZl-CKB infected cells in one single step involving an ion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.