Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.
Characterization of a functional recombinant human creatine kinase-MB isoenzyme prepared by tandem affinity purification from Escherichia coli.
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通过串联亲和纯化从大肠杆菌中制备的功能性重组人肌酸激酶-MB 同工酶的表征。
DOI：
10.1007/s00253-017-8286-5
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发表时间：
2017
影响因子：
5
通讯作者：
Xiao Fei
中科院分区：
文献类型：
--
作者：
Zou Lihui;Su Wen;Wang Meng;Huang Wei;Zhao Haijian;Zhang Enyi;Jin Junhua;Xu Hongtao;Xiao Fei
肌酸激酶同工酶CK-MB作为心肌损伤的生物标志物已被广泛应用。虽然临床实验室已经使用了各种方法来测量CK-MB的活性或质量，但需要一个CK-MB标准来消除方法间的偏差。由于体外表达的人肌酸激酶会产生CK-MM、CK-MB和CK-BB三种同工酶，因此建立一种有效的方法从混合物中分离纯化CK-MB具有重要意义。本研究旨在利用串联亲和纯化技术(TAP)纯化重组CK-MB蛋白，并评价其临床应用价值。合成编码CK-manCK-B的优化序列后，与TAP标签(6His和SBP)结合，插入pRSFDuet载体，构建成6His-CK-M-SBP-CK-B-prsf表达载体，转化大肠杆菌BL21(DE3)进行表达。TAP后，获得了纯化的CK-MB蛋白。我们还用不同浓度的工程化CK-MB和标准CK-MB(Randox)进行了回收试验，结果表明，工程化CK-MB可以作为参照物。此外，对重组CK-MB的稳定性研究表明，重组CK-MB蛋白在−80℃下长期保存具有较高的稳定性。结论：TAP纯化的重组CK-MB蛋白可能是一种更好、更便宜的临床实验室标准或参考物质。
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 codingCK-MandCK-Bwere 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 intoEscherichia coliBL21 (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.