era基因的高表达

Overexpression of the era Gene

为了高表达产生Era蛋白,借助计算机从Gene Bank中寻找N端几个氨基酸序列与Era相同的蛋白质,选其中能在大肠杆菌内高表达的λ Ea8.5蛋白,以其基因5’端序列替代天然era基因5’端序列,从而赋予era基因转录物以强的翻译起始信号,而不改变其编码的氨基酸序列。将此重组era基因置于P_L启动子下游、构建得质粒pCE31,引入大肠杆菌TAP106,经诱导后大量合成Era蛋白,且其他蛋白质的合成显著被抑制,从而使Era的含量能超过菌体总蛋白量的80%。经简单裂菌、洗涤步骤,就获得具有能特异结合鸟苷酸活性的、电泳单带纯的Era蛋白。

For overproduction of Era protein, λ ea8.5 gene have been chosenfrom gene bank because of Ea8.5 protein has sequence of several aminoacids of its N-terminal identical to tha of Era and it can be highlyexpressed in E.coli. 5' end sequences of era gene was substituted bysynthetic oligonucleotide which sequence is identical to that of 5' endof ea8.5 gene, so that endowed the transcripts of era gene with strongtranslational initiation signal and without changing the amino acid se-quence of its translational product. Plasmid pCE31, which harbors therecombined era gene under the control of P_L Promoter could expressvery high levels of Era protein and other cellular protein synthesis wasnearly shutoff during the period of induction, resulting Era constitutedover 80% of total cell protein, Electrophoretic pure Era protein withspecific guanine nucleotide-binding activity was obtained by simpleprocedure including lyzing the cells and washing the pellet of the lysate.