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Expression, Purification of Human gAd in E.coli and Identification of Its Biological Activity |
ZHANG Li-jian1, SUN Lu1, GUO Yun-ping1, LI Dong-dong1, WANG Zeng-lu2, LIU Yi1, TAO Ling1 |
1. Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China;
2. Biotechnology Center, School of Pharmacy, The Fourth Military Medical University, Xi’an 710032, China |
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Abstract Objective: To construct the prokaryotic expression vector of human gAd gene and obtain the no-tagged recombinant human gAd protein. Methods: Total RNA was extracted from human adipose tissue. The gAd cDNA was obtained with RT-PCR technique and subcloned into a prokaryotic exprssion vector pET-22b(+) to generate pET-22b(+)-gAd. The expression of no-tagged recombinant human gAd protein was induced by IPTG in E.coli BL21(DE3). The gAd protein produced as inclusion bodies at an elevated level. The inclusion bodies were solubilized by alkaline shock, and then refolded and purified after acetone precipitation. SDS-PAGE and Western blot were used for identification of the purified gAd. The abilities of gAd to induce the phosphorylation of AMPK in HUVECs and to protect hearts after myocardial ischemia/reperfusion in mice were used for its biological activity assay. Results: The human gAd coding sequence was correctly cloned into pET-22b(+) vector. After expression and purification, the recombinant human gAd significantly induced the phosphorylation of AMPK in HUVECs and protected hearts from myocardial ischemia/reperfusion injury. Conclusion: The no-tagged recombinant human gAd was successfully expressed and purified in prokaryotic expression system with high biological activity.
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Received: 22 November 2012
Published: 25 June 2013
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Cite this article:
ZHANG Li-jian, SUN Lu, GUO Yun-ping, LI Dong-dong, WANG Zeng-lu, LIU Yi, ZHANG Ying-qi, TAO Ling. Expression, Purification of Human gAd in E.coli and Identification of Its Biological Activity. China Biotechnology, 2013, 33(6): 68-73.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2013/V33/I6/68
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