Mechanism of high level expression of foreign gene caused by pHsh vector in Escherichia coli

Abstract:

pHsh is a novel high level expression vector of Escherichia coli, in which the regulatory promoters are recognized by the 32-kD sigma factor (σ32). In normal E. coli cells, the total time length of heat-shock response is about 12 min, however, in E. coli cells carrying recombinant high-copy pHsh vectors, the heat-shock response can sustain 4?10 h. In order to understand the mechanism of hign level expression of foreign gene in E. coli carrying pHsh vector, we employed xynIII gene encoding a xylanase as the representative of foreign genes. Firstly, the effect of copy-number of pHsh on the expression level of xynIII gene was tested, then the difference of the concentration of σ32 between in the E. coli cells harboring pHsh-xynIII and in the E. coli cells harboring pLac-xynIII was assayed by using western-blot under either inducing (30 °C→42 °C) or non-inducing conditions (30 °C). Finally, under different temperatures, the heat-shock level at steady state in recombinant E. coli cells harboring pHsh-xynIII was evaluated by the xylanase activity. the results showed that the high expression level of foreign genes in pHsh should attribute to the following three aspects: high-copy-number of pHsh enhanced the foreign gene dosage accessible for expression and leaded to a high productivity; Owing to the present of the pHsh, σ32 level in E. coli cells was significantly higher than that in E. coli cells without pHsh, thus the heat-shock level was significantly enhanced in E. coli cells harboring pHsh; The considerable heat-shock level at steady state in recombinant E. coli cells harboring pHsh was helpful for high level expression of foreign genes.

Key words:Escherichia coli, pHsh vector, Heat-shock, Sigma32, Expression