中国生物工程杂志

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.