Abstract
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.
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This work was supported by the National Natural Science Foundation of China (Grant 81400036 and Grant 81571384).
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Zou, L., Su, W., Wang, M. et al. Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli . Appl Microbiol Biotechnol 101, 5639–5644 (2017). https://doi.org/10.1007/s00253-017-8286-5
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DOI: https://doi.org/10.1007/s00253-017-8286-5