RESUMO
Objective To construct a prokaryotic expression system for human creatine kinase(CK) MM isozyme,purify the recombinant protein of CK expressed in Eschericheia coli(E.coli) and examine the stability of the recombinant protein for its application in CK measurement system as the quality control.Methods Total RNA of extracted from fetal cardiac muscle was reversetranscripted and cDNA encoding human CK was amplified which was inserted into pET-15b plasmid vector.The recombinant plasmid was transfered into E.coli BL21(DE3) and induced by isopropyl-?-D-thiogalactopyranoside(IPTG).Recombinant CK-MM was separated from bacterial proteins by affinity chromatography on a Ni2+-Sepharose column.The activity of the recombinant enzyme was observed in different matrixes.Results The enzymatic activity of the crude extracts of CK-MM was up to 280,000U/L.After one step affinity chromatography,the fusion protein showed a single band in SDS-PAGE gel.The purified protein was stable and the enzymatic activity remained unchanged for a month in the matrix containing bovine serum albumin,EDTA and 1,4-Dithiothreitol(DTT).Conclusion The recombinant CK-MM showed key properties of the native creatine kinase isozyme and may be used as control and calibrator for determination of serum CK-MM.