Expression,Purification of PUMA-BH3 Death Domain Peptide in E.coli and Identification of Its Pro-apoptotic Activity
- VernacularTitle:PUMA-BH3结构域短肽的原核表达、纯化及促凋亡活性鉴定
- Author:
Yu-Wen ZHANG
;
Xing-Han LIU
;
Hui-Min LIN
;
Ji-Hong LI
;
Hong-Xing MA
;
Yuan-Li LIU
;
- Publication Type:Journal Article
- Keywords:
BH3 death domain peptide of PUMA Recombinant expression Mitochondria Pro-apoptotic activity
- From:
China Biotechnology
2006;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.