High level prokaryotic expression of neuron protective protein TAT-Bcl-XL and preliminary detection of its anti-apoptosis activity
- VernacularTitle:神经元保护蛋白TAT-Bcl-XL的体外高效表达及其功能的初步研究
- Author:
Jianping DING
;
Jianping JIA
- Publication Type:Journal Article
- Keywords:
Protein transduction domain;
Apoptosis;
Ischemic brain damage
- From:
Chinese Journal of Immunology
1985;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To prepare the recombinant neuron protective protein TAT-Bcl-XL in E.coli expression system and to determine its anti-apoptosis activity.Methods:DNA fragment of TAT-Bcl-XL was obtained by RT-PCR using primers that were specific for Bcl-XL gene. Prokaryotic expression vector(pTBTOPO) was constructed by insertion of TAT-Bcl-XL DNA fragment into pCRT7/CT-TOPO vectors, and the recombinant protein was detected by SDS-PAGE and Western blot analysis using anti-V5 tag epitope antibody as the primary antibody. The recombinant fusion protein was purified by affinity chromatography, and the anti-apoptosis function of the purified protein was determined by flow cytometry.Results:A molecular weight of 30 000 protein was detectable in both SDS-PAGE and Western blot analysis. Immunofluorescent staining showed the fusion protein was distributed in cell plasma after incubation with 200 nmol/L fusion protein. Flow cytometry data indicated that the recombinant protein could enhance cell survival by 40% when 293T cells were treated with 250 ?mol/L zinc chloride as a supplementation in cell culture media.Conclusion:High level expression of the reported neuron protective protein was obtained in E.coli expression system, and the purified recombinant proteins remained its anti-apoptosis activity in our preliminary characterization.
