大肠杆菌精氨酰tRNA合成酶基因的诱导高表达

在高表达大肠杆菌精氨酰ｔＲＮＡ合成酶基因５５０倍的基础上，将ａｒｇ２的编码起始位点经基因突点突变导入ＮｃｏＩ限制性内切酶的位点后，重组到受异丙基硫代－β－Ｄ半乳糖苷诱导的ｐＴｒ９９Ｂ质粒上，使ａｒｇＳ比受体菌表达高近２０００倍。通过一步ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析则可得到ＳＤＳ－ＰＡＧＥ一条带的ＡｒｇＲＳ，比活为１５０００ｕ／ｍｇ，与文献相同。

Study on the Overexpression of the Gene Encoding Arginyl tRNA Synthetase under Induction

Arginyl tRNA synthetase(ArgRS) from E.coli was overproduced from transformant containing the gene encoding this enzyme( arg S) by 550 fold higher than that from host cell. By site directed mutagenesis the cleavage site for Nco I restriction endonuclease was introduced into the start codon of arg S. The mutant gene was recombinated with plasmid pTrc99B under IPTG control. In the transformant containing the recombinated plasmid, arg S can overexpressed 2 000 times higher than that in host cell. Through one step DEAE Sepharose column chromatography, ArgRS was purified to be one band by SDS PAGE and its specific activity was 15 000 u/mg, similar to reported values. The mutant ArgRS2ND which had the second residue asparagine replaced by aspartic acid showed no change of activity, kinetic constants nor the thermal and denaturational stabilities.