Production of native creatine kinase B in insect using a baculovirus expression vector
نویسندگان
چکیده
A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZl-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal condi tions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chro matography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZlCKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed pro tein had a specific activity of 239 U/mg protein. (Mol Cell Biochem 143: 59-65, 1995)
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