%A ZHANG Qi, WU Jia-wei, ZHOU Ai-wu %T Preparation and activity identification of human cyclophilin B %0 Journal Article %D 2016 %J Journal of Shanghai Jiao Tong University (Medical Science) %R 10.3969/j.issn.1674-8115.2016.12.008 %P 1723- %V 36 %N 12 %U {https://xuebao.shsmu.edu.cn/CN/abstract/article_11382.shtml} %8 2016-12-28 %X

Objective · To construct an expression vector for human cyclophilin B (hCypB) and prepare active CypB via the prokaryotic expression system. Methods · The DNA sequence enconding hCypB was amplified by PCR and inserted into expression vector pGEX-6P-1 by double enzyme digestion. hCypB was further purified with affinity chromatography and the activity of over-expressed hCypB was verified with GST-pulldown. Results · The recombinant CypB expression plasmid was successfully constructed and verified with DNA sequencing. GST-CypB fusion protein was purified to a purity of 90% by affinity chromatography with a yield of 26 mg of fusion protein from a liter of overnight E. coli culture. GST-pulldown further confirmed that this GST-hCypB fusion protein could form the ternary complex with prolyl 3-hydroxylase 1 (P3H1) and cartilage-associated protein (CRTAP). Conclusion · Large amount of active and highly purified hCypB can be obtained via this experiment method within short time period.