摘要
目的:研究高温条件下小分子热休克蛋白IbpB在大肠杆菌体内的客户蛋白以及在此温度下IbpB对其客户蛋白的作用。方法构建表达IbpB蛋白的重组质粒pBAD-ibpB,pBAD质粒为对照,转化到BW25113-ΔibpB菌株。转化pBAD-ibpB质粒的BW25113-ΔibpB菌株在45℃热处理之后加入甲醛进行交联,亲和层析法对交联产物进行纯化,质谱法对捕获的客户蛋白进行鉴定,过氧化氢酶活性测定和蛋白热聚集体分离的方法检测IbpB对其客户蛋白的作用。结果质谱鉴定结果表明IbpB在高温条件下互相作用的客户蛋白主要是过氧化氢酶KatG、色氨酸降解酶TnaA和延伸因子EF-Tu;在ibpB敲除株中回补IbpB可以减少过氧化氢酶在高温条件下的活性丧失,可以阻止TnaA和EF-Tu在高温条件下发生的热聚集。结论 KatG、TnaA和EF-Tu是IbpB在高温条件下相互作用的主要客户蛋白,IbpB在高温条件下对上述客户蛋白具有明显的保护作用。
Objective To investigate the client proteins of small heat shock protein IbpB in Escherichia coli cells under a high temperature and the effect of IbpB on its client proteins at this temperature.Methods A recombinant vec-tor pBAD-ibpaB was constructed which could express IbpB protein, while the vector pBAD was used as a control.Both vectors were transfected into Escherichia coli BW25113-ΔibpB cell and then heated at 45℃ before formaldehyde cross-linking.The resultant products were purified by affinity chromatography, and the client proteins of interest were identified by mass spectrometry.Finally, the effects of IbpB on these client proteins under a high temperature were determined by catalase activity assay and protein thermal aggregation isolation.Results According to mass spectrometry, the client proteins interacting with IbpB under a high temperature included catalase KatG, tryptophanase TnaA and elongation factor EF-Tu.The addition of IbpB in ibpB-knock out strains could reduce heat-induced catalase inactivation and prevent heat-induced aggregation of TnaA and EF-Tu.Conclusion The main client proteins interacting with IbpB under a high temperature are KatG, TnaA and EF-Tu, which can obviously protect by IbpB at this temperature.
出处
《徐州医学院学报》
CAS
2015年第12期841-844,共4页
Acta Academiae Medicinae Xuzhou
基金
江苏省自然科学基金青年基金(BK20130232)