文献类型：期刊文献

英文题名：High-level expression, purification and pro-apoptosis activity of HIV-TAT-survivin (T34A) mutant to cancer cells in vitro

作者：Ma, Xingyuan[1]; Zheng, Wenyun[1,2]; Wei, Dongzhi[1]; Ma, Yushu[1]; Wang, Tianwen[1]; Wang, Jinzhi[1]; Liu, Qinghai[1]; Yang, Shengli[1,3]

机构：[1]E China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shenyang Pharmaceut Univ, Sch Pharmaceut Engn, Shenyang 110016, Peoples R China;[3]Chinese Acad Sci, Shanghai Res Ctr Biotechnol, Shanghai 200233, Peoples R China

年份：2006

卷号：123

期号：3

起止页码：367

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：2006199865085);WOS:【SCI-EXPANDED(收录号:WOS:000237881900011)】；

语种：英文

外文关键词：TAT-survivin (T34A) mutant; high-level expression and purification; pro-apoptosis

摘要：Survivin, a novel member of the IAP family, was observed to express in the most common human cancers. Anti-cancer therapy targeting survivin had drawn considerable attention. This study focused on high-level expression of recombinant protein TAT-survivin (T34A) mutant in E. coli, purification and bioactivity of pro-apoptosis to various cell lines in vitro. The cDNA encoding survivin was cloned by RT-PCR from breast cancer cell lines B-cap37. After PCR site-directed mutagenesis and construction of expression vector pRSET-B-TAT-survivin (T34A), targeted TAT-survivin (T34A) protein was expressed highly in E. coli BL21 (DE3) by 0.5 mM IPTG induction and its yield could reach 650 mg/l in fermentation culture. The fusion protein in a form of inclusion body was then solubilized, refolded and purified to a purity of 98% by cation exchange chromatography and size-exclusion chromatography. Four hundred and eighty milligrams protein of interest was obtained in per liter fermentation culture. This showed that the efficient procedures of large-scale expression and purification were successful for the mass production of the recombinant protein. Pro-apoptosis effects of target protein on four cancer cell lines and one normal cell line from human were confirmed by the change of morphology, and pro-apoptosis activity was evaluated by MTT, fluorescent staining of nuclei and flow cytometry assay. Results indicated that B-cap37 and SW1990 were very sensitive to TAT-survivin (T34A) protein. This finding revealed the recombinant protein was promising as an anti-cancer drug. (c) 2005 Elsevier B.V. All rights reserved.