[The cloning and expression of angiogenic inhibitor tumstatin(45-132) in E. coli]
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Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Published on: January 22, 2014
Yi-qin Luo1, Liang-hua Wang, Yi Qiu
1Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China.
Aim:
To clone tumstatin(45-132) gene and to express recombinant human tumstatin(45-132) in E. coli BL21.
Methods:
Tumstatin gene was cloned by RT-PCR and then tumstatin(45-132) gene amplified from tumstatin gene was cloned into pBV220. The recombinant plasmid pBV220-tumstatin(45-132) was sequenced and transformed into E.coli BL21. E. coli BL21 transformed with the recombinant plasmid pBV220-tumstatin(45-132) was induced at 42 degrees Celsius. After the recombinant tumstatin(45-132) was purified, its bioactivity was detected by endothelia cell proliferation test.
Results:
264 bp tumstatin(45-132) fragment was cloned and its sequence was identical with that in GenBank. The tumstatin(45-132) was expressed in E. coli BL21. Expressed product accounted for about 10% of total bacterial proteins and its relative molecular mass (M(r)) was 9,600. The purified protein showed inhibitory effect on proliferation of endothelia cells ECV304 in vitro.
Conclusion:
tumstatin(45-132) gene has been cloned and expressed successfully in E.coli BL21. Expressed tumstatin(45-132) can inhibit the proliferation of endothelial cell ECV304.
