Cloning and expression of functional rabbit muscle creatine kinase in Escherichia coli. Addressing the problem of microheterogeneity.
The gene encoding rabbit muscle creatine kinase (CK) has been subcloned into a single plasmid, and the protein expressed in a soluble and functional form in Escherichia coli. The amino terminus, specific activity, and electrophoretic mobility of the E. coli-expressed creatine kinase are all identical with that of creatine kinase purified from rabbit skeletal muscle. Surprisingly, isoelectric focusing shows that the expressed protein displays no less heterogeneity than the tissue-purified material. The identification of the source(s) of this heterogeneity is important for the preparation of highly homogeneous material needed for structural studies and clinical applications. This issue also has implications for studies of the developmental regulation and tissue localization of the various CK genes. Our results allow us to eliminate some of the proposals, such as the presence of multiple alleles, alternative ribosomal initiation sites, and post-translational glycosylation or phosphorylation that have been suggested to explain the presence of the numerous isoforms present in apparently pure preparations of CK.
Chen
L H
LH
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Babbitt
P C
PC
Vásquez
J R
JR
West
B L
BL
Kenyon
G L
GL
D000818:Animals; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D007525:Isoelectric Focusing; D009132:Muscles / Q000201:enzymology*; D010957:Plasmids; D011817:Rabbits
D003402:Creatine Kinase