Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli.
Sarcomeric mitochondrial creatine kinase (Mib-CK) of chicken was expressed in Escherichia coli as a soluble enzyme by using an inducible phage-T7 promoter. Up to one third of the protein in E. coli extracts consisted of soluble recombinant Mib-CK in an enzymically active form. Approx. 20 mg of nearly-homogenous Mib-CK was isolated in a two-step isolation procedure starting with 1 litre of isopropyl beta-D-thiogalactopyranoside-induced E. coli culture, whereas previous attempts to express other CK genes in E. coli have resulted in 20-fold lower yields and inclusion-body formation. Selection of the Mib-CK expression plasmid on media containing kanamycin rather than ampicillin extended the time period of maximal Mib-CK expression. Recombinant Mib-CK displayed an identical N-terminal amino acid sequence, identical Km for phosphocreatine and Vmax. values, the same electrophoretic behaviour and the same immunological cross-reactivity as the native enzyme isolated from chicken heart mitochondria. The recombinant Mib-CK had the same molecular mass as native chicken Mib-CK in m.s. analysis, indicating that post-translational modification of the enzyme in chicken tissue does not occur. As judged by gel-permeation chromatography and electron microscopy, recombinant enzyme formed predominantly octameric oligomers with the same overall structure as the chicken heart enzyme. Furthermore, the enzymes isolated from both sources formed protein crystals of space group P42(1)2, when grown in the absence of ATP, with one Mi-CK octamer per asymmetric unit. The indistinguishable X-ray-diffraction patterns indicate identical structures for the native and recombinant proteins.
Furter
R
R
Institute for Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
Kaldis
P
P
Furter-Graves
E M
EM
Schnyder
T
T
Eppenberger
H M
HM
Wallimann
T
T
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology; D002645:Chickens; D003402:Creatine Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D003402:Creatine Kinase; D012321:DNA-Directed RNA Polymerases