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

Paper ID Volume ID Publish Year Pages File Format Full-Text
25611 43584 2006 12 PDF Available
Title
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 0.5 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.

Keywords
TAT-survivin (T34A) mutant; High-level expression and purification; Pro-apoptosis
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High-level expression, purification and pro-apoptosis activity of HIV-TAT-survivin (T34A) mutant to cancer cells in vitro
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 123, Issue 3, 29 May 2006, Pages 367–378
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us