|
|
|
| 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 |
|
|
|
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.
|
|
Received: 22 November 2012
Published: 25 June 2013
|
|
|
|
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
|
|
|
| [1] Wei Gu,Yang Li.The therapeutic potential of the adiponectin pathway. Biod Rugs, 2012,26 (1): 1-8.
[2] Siaso S, Tousoulis D, Kollia C, et al. Adiponectin and cardiovascular disease: mechanisms and new therapeutic approaches. Current Medicinal Chemistry, 2012, 19, 1193-1209.
[3] Berg A H, Combs T P, Scherer P E, et al. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nature Medicine, 2001, 7: 947-953.
[4] Yamauchi T, Kamon J, Waki H, et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nature Medicine,2001,7: 941-946.
[5] Ling T, Erhe G, Xiang Y J,et al.Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress. Circulation, 2007,115:1408-1416.
[6] Scherer P E,Williams S,Fogliano M, et al.A novel serum protein similar to Clq produced exclusively in adipocytes. Journal of Biological Chemistry,1995,270(45):26746-26749.
[7] Fruebis J, Tsao T S, Javorschi S, et al.Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proceedings of The National Academy of Sciences of The United States of America, 2001,98(4):2005-2010.
[8] Yamauchi T, Kamon J, Minokoshi Y, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.Nature Medicine,2002,8(11):1288-1295.
[9] Arita Y, Kihara S, Ouchi N,et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochemical and Biophysical Research Communications, 1999,257:79-83.
[10] Hotta K Funahashit,Bodkin N L. Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys.Diabetes, 2001,50:1126-1133.
[11] Kadowaki T, Yamauchi T, Kubota N.Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. Journal of Clinical Investigation, 2006,116:1784-1792.
[12] Liu D G, Liu H L, Song T J,et al. Functional expression of the globular domain of human adiponectin in Pichia pastoris.Biochemical and Biophysical Research Communications, 2007,363:769-775.
[13] 王云龙, 李永超, 昌静峰, 等. 人脂联素原核表达纯化及活性检测. 动物医学进展, 201l,32(10):36-39. Wang Y L,Li Y C,Chang J F, et al.Prokaryotic expression and purification of human adiponetin and its immunological activity detection.Progress in Veterinary Medicine,2011,32(10):36-39.
[14] Hong F G,Yu M X, Bryan L,et al.Generation of novel long-acting globular adiponectin molecules. Journal of Molecular Biology, 2010,399:113-119.
[15] Heiker J T, Klöting N, Blüher M,et al.Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization. Biochemical and Biophysical Research Communications, 2010,398:32-37. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
