热休克转录因子（σ^（32））基因的克隆及其在大肠杆菌中的表达

GENE CLONING AND EXPRESSION OF HEAT SHOCK TRANSCRIPTIONAL FACTOR σ 32 IN E.COLI

用ＰＣＲ方法获得大肠杆菌热休克蛋白转录因子σ３２的编码基因ｒｐｏＨ，并将其克隆至含有Ｐｔａｃ启动子的表达载体ｐＵＨＥ中。经ＩＰＴＧ诱导，在ｒｐｏＨ缺陷菌株ＳＧ１３００９中高效表达Ｃ－端融合有６个组氨酸的σ３２，表达量为菌体总蛋白的７．６％。３５Ｓ细胞内掺入实验表明：即使在较低的温度下，表达产物σ３２－（Ｈｉｓ）６也能导致热休克蛋白如ＧｒｏＥＬ、ＤｎａＫ。

When cells and organisms are suddenly exposed to a high temperature, a small set of proteins, known as heat shock proteins, are transiently induced. These proteins are very important in the physiological and biochemical process; their functions appear not only in heat shock and other stress responses, but also in the normal growth of cells. For example, many of them are involved in the folding and resemby of proteins. In E. coli, there are about 20 heat shock proteins, whose expression is positively regulated by the he at shock transcriptional factor σ 32 . σ 32 is the product of E.coli rpoH gene, with molecular weight of 32KDa. It binds with α 2ββ’ (core enzyme) to form the RNA polymerase holoenzyme (Eσ 32 ), recognizes the promoter of heat shock proteins, and regulates the synthesis of heat shock proteins at the level of mRNA transcription. In this experiment, the rpoH gene was obtained by PCR and cloned into pUHE plasmid with tac promoter. Upon induction with IPTG, it directed the expression of recombinant σ 32 tailed with 6 additional histidine residues at its C-termini. The level of expression is about 7.6 percent of the cellular total proteins. Radiolabeling in vivo with 35 S experiments show that the over expression of σ 32 -6His can apparently increase the synthesis of the heat shock proteins such as GroEL, DnaK HtpH even at low temperature such as 30℃.