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

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.
AuthorsXingyuan Ma, Wenyun Zheng, Dongzhi Wei, Yushu Ma, Tianwen Wang, Jinzhi Wang, Qinghai Liu, Shengli Yang
JournalJournal of biotechnology (J Biotechnol) Vol. 123 Issue 3 Pg. 367-78 (May 29 2006) ISSN: 0168-1656 [Print] Netherlands
PMID16406157 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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
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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