High-level expression, purification and pro-apoptosis activity of HIV-TAT-survivin (T34A) mutant to cancer cells in vitro.
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 0.5mM 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.
Ma
Xingyuan
X
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Zheng
Wenyun
W
Wei
Dongzhi
D
Ma
Yushu
Y
Wang
Tianwen
T
Wang
Jinzhi
J
Liu
Qinghai
Q
Yang
Shengli
S
D000970:Antineoplastic Agents / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000008:administration & dosage* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015696:Gene Products, tat / Q000008:administration & dosage* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009369:Neoplasms / Q000188:drug therapy* / Q000473:pathology*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000378:metabolism; D000077022:Survivin; D054322:tat Gene Products, Human Immunodeficiency Virus
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D015696:Gene Products, tat; C512124:HIV-TAT-survivin (T34A) protein; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000077022:Survivin; D054322:tat Gene Products, Human Immunodeficiency Virus