表达载体pHsh对大肠杆菌热休克系统中负调控机制的影响

pHsh是一种由σ32识别和启动外源基因表达的新型高效的大肠杆菌表达载体。正常E.coli细胞在热激诱导条件下，σ32的浓度在5 min内到达高峰，随后被3个负调控蛋白Dnak、DnaJ、GrpE结合导致失活或降解，整个热休克反应持续约12min。在携带有外源基因的高拷贝pHsh 的E.coli细胞中，外源基因却能持续高效表达4～10 h，这一现象表明了此时细胞中的σ32比没有携带质粒的细胞内σ32的浓度要高。σ32浓度的增高有可能是由于3个负调控蛋白Dnak、DnaJ、GrpE在细胞内的含量比正常情况下降低的结果。为了验证这一推测，从E.coli中克隆了Dnak、DnaJ、GrpE的编码基因，表达并初步纯化了其重组蛋白以作分子标记，采用双向电泳技术，分析携带质粒（pHsh+）和不携带质粒的E.coli(pHsh-)细胞在热休克后胞内蛋白质组的差异。该项实验通过与检索到的标准的E.coli蛋白质组图谱进行比较鉴别出的两个蛋白Dnak、GrpE，并通过对比目标点的大小和深浅发现pHsh+中的Dnak均少于pHsh─中的目标蛋白，所得结果与上述假设一致。

The Effect of Expression Vector pHsh on Negative Regulatory Mechanism of Heat-shock System in Escherichia coli

pHsh is a novel high level expression vector of Escherichia coli, in which the regulatory promoters are recognized by the 32-kD sigma factor ( 32). The concentration of 32 protein in E.coli cells without pHsh vector peaks at about 5 min after a temperature shift from 30℃ to 42℃, and then declines rapidly to a very low level because of its degradation and inactivation caused by interaction between three negative regulatory proteins-DnaK, DnaJ, GrpE and 32, the whole process is about 12 min. In E.coli cells harboring recombinant high-copy pHsh vectors, the heat-shock response can sustain 4～10 h, which indicates the concentration of 32 protein was increased compared to that in E.coli cells without pHsh vectors. The increasing of the concentration of 32 is most likely due to the decreasing of the concentration of DnaK, DnaJ and GrpE. In this paper, the changes of the three negative regulatory proteins in Escherichia coli proteome caused by the existence of the pHsh expression vector were assayed by two-dimensional electrophoresis. The genes encoding the three negative regulative proteins were cloned into pET vectors and overexpressed. After the resulting recombinant proteins were partially purified, they acted as molecular markers in order to identify their positions in 2-D gels. After thermal induction and referring to standard 2-D gel map in SWISS-2DPAGE database, the proteomes of E.coli cells harboring pHsh-xynIII vectors obtained from different periods were analyzed by two-dimensional gel electrophoresis and compared to those of E.coli cells without pHsh-xynIII, and the DnaK and GrpE were detected in 2-D gels, the data showed the DnaK protein in pHsh+ cells was obviously decreased compared to that in cells without pHsh, therefore the result verified the foresaid hypothesis.