EXPRESSION OF ACTIVE OCTAMERIC CHICKEN CARDIAC MITOCHONDRIAL CREATINE-KINASE IN ESCHERICHIA-COLI

Sarcomeric mitochondrial creatine kinase (Mi(b)-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 Mi(b)-CK in an enzymically active form. Approx. 20 mg of nearly-homogenous Mi(b)-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 ''press other CK genes in E. coli have resulted in 20-fold lower yields and inclusion-body formation. Selection of the Mi(b)-CK expression plasmid on media containing kanamycin rather than ampicillin extended the time period of maximal Mi(b)-CK expression. Recombinant Mi(b)-CK displayed an identical N-terminal amino acid sequence, identical K(m) for phosphocreatine and V(max) values, the same electrophoretic behaviour and the same immunological cross-reactivity as the native enzyme isolated from chicken heart mitochondria. The recombinant Mi(b)-CK had the same molecular mass as native chicken Mi(b)-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.