关键词: 热休克蛋白70；纯化；Triton X-114；内毒素类

Abstract:

Abstract:Objective
To express and purify the recombinant BCG heat shock protein 70 (BCG HSP70),and remove endotoxin from it.Methods E.coli. BL21(DE3) containing the recombinant plasmid of pET28a/HSP70 (pET28a/HSP70/BL21) was induced with IPTG in 10 L fermentor,and the expression of BCG HSP70 was detected by SDS-PAGE. Then the bacteria were disrupted by sonication. BCG HSP70 was purified by successive applications of Nikel-affinity chromatography on Sepharose 4B,TritonX-114 washing,gel filtration on Sephadex G-25 and ion-exchange chromatography on Q-Sepharose FF.The purified BCG HSP70 was identified by SDS-PAGE.The concentration of the purified BCG HSP70 was detected by Lowry assay.And the purified BCG HSP70 was incubated in the 37 ℃ water bath for 0 to 4 h to analyze its stability.Endotoxin in the purified proteins was determined by Limulus amebocyte lysate assay.Results 96 g wet weight of bacterial pellet was obtained after 3 h of induction of pET28a/HSP70/BL21 in the fermentor,and the protein with relative molecular mass of 70 000 approximately accounted for about 29.6% of the total bacterial protein.The protein with relative molecular mass of 70 000 was purified to 96.5% purity,and the concentration of the purified protein was 1.2 g•Ｌ
^-1.When incubated in the 37 ℃ water bath for 4 h,the BCG HSP70 accounted for approximately 95.1% of the total protein.Endotoxin in the purified protein was less than 0.01 EU?•μg^-1.Conclusion The recombinant BCG HSP70 is expressed and purified successfully,the endotoxin in the purified BCG HSP70 is removed effectively.

Key words: heat shock protein 70;purification；Triton X-114；endotoxins