Abstract |
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.
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Authors | Xingyuan Ma, Wenyun Zheng, Dongzhi Wei, Yushu Ma, Tianwen Wang, Jinzhi Wang, Qinghai Liu, Shengli Yang |
Journal | Journal of biotechnology
(J Biotechnol)
Vol. 123
Issue 3
Pg. 367-78
(May 29 2006)
ISSN: 0168-1656 [Print] Netherlands |
PMID | 16406157
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Antineoplastic Agents
- Apoptosis Regulatory Proteins
- Gene Products, tat
- HIV-TAT-survivin (T34A) protein
- Recombinant Fusion Proteins
- Recombinant Proteins
- Survivin
- tat Gene Products, Human Immunodeficiency Virus
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Topics |
- Antineoplastic Agents
(administration & dosage)
- Apoptosis
(drug effects)
- Apoptosis Regulatory Proteins
(administration & dosage, genetics, isolation & purification, metabolism)
- Cell Line, Tumor
- Escherichia coli
(genetics, metabolism)
- Gene Products, tat
(administration & dosage, genetics, isolation & purification, metabolism)
- Humans
- Mutagenesis, Site-Directed
- Neoplasms
(drug therapy, pathology)
- Recombinant Fusion Proteins
- Recombinant Proteins
(administration & dosage, isolation & purification, metabolism)
- Survivin
- tat Gene Products, Human Immunodeficiency Virus
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