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 (AcDZ1-BCK). Sf9 cells infected with this recombinant 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 filtration chromatography 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 AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.
de Kok
Y J
YJ
Department of Cell Biology and Histology, Medical Faculty University Nijmegen, The Netherlands.
Geurds
M P
MP
Sistermans
E A
EA
Usmany
M
M
Vlak
J M
JM
Wieringa
B
B
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003402:Creatine Kinase / Q000096:biosynthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D003402:Creatine Kinase