Overexpression, purification, and preliminary X-ray crystallographic analysis of human brain-type creatine kinase

Abstract

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transfered through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at 22 degrees C using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to 2.2 angstrom resolution using synchrotron radiation. The crystals belonged to the tetragonal space group P4(3)2(1)2, with cell parameters of a=b=97.963, c=164.312 angstrom, and alpha= beta=gamma=90 degrees. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass (V-m) of 1.80 angstrom(3) Da(-1) and a solvent content of 31.6%.