甲醛交联法研究高温条件下大肠杆菌体内小分子热休克蛋白IbpB的客户蛋白

Research on the client proteins of small heat shock protein IbpB in Escherichia coli cells under a high temperature by formaldehyde cross-linking

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.