Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli

Appl Microbiol Biotechnol. 2017 Jul;101(14):5639-5644. doi: 10.1007/s00253-017-8286-5. Epub 2017 Apr 21.


Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

Keywords: CK-MB; Enzyme activity; Gene recombination; Protein expression; Tandem affinity purification.

Publication types

  • Evaluation Study

MeSH terms

  • Chromatography, Affinity / economics
  • Chromatography, Affinity / methods*
  • Clinical Enzyme Tests
  • Creatine Kinase, MB Form / genetics*
  • Creatine Kinase, MB Form / isolation & purification
  • Creatine Kinase, MB Form / metabolism*
  • Enzyme Stability
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Humans
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Reference Standards


  • Recombinant Proteins
  • Creatine Kinase, MB Form