大肠杆菌JM83精氨酰－tRNA合成酶基因的克隆、测序及表达

用聚合酶链反应（ＰＣＲ）以大肠杆菌ＪＭ８３基因组ＤＮＡ为模板，扩增了精氨酰－ｔＲＮＡ合成酶（ＡｒｇＲＳ）基因。将该基因重组到载体ｐＵＣ１８上转化到大肠杆菌ＴＧ１中，得到在转化子中ＡｒｇＲＳ的高表达。粗抽液中ＡｒｇＲＳ的氨酰化活力，ＴＧ１和ＴＧ１转化子分别为１．６５Ｕ／ｍｇ和２１０Ｕ／ｍｇ，后者为前者的１２７倍。ＤＮＡ顺序测定表明，与从大肠杆菌ＪＡ２００中克隆到的ＡｒｇＲＳ基因相比９１３位碱基为Ａ而不为Ｃ，这种变化使ＡｒｇＲＳ的３０５位氨基酸残基由Ｇｌｎ变为Ｌｙｓ，但这种改变不影响酶的活力。

Cloning, Sequencing and Expreesion of the Gene of Arginyl-tRNA Synthetase from E.coli JM83

The gene of arginyl-tRNA synthetase(ArgRS) was amplified from the genomic DNA of E.coli JM83 by PCR, ligated into pUC18 and transformed into E. coli TGl. ArgRS was overproduced in TG1 transformat. The specific activity of aminoacylation of ArgRS in the crude extract of TG1 and TG1 transformat containing recombinant plasmid was 1. 65 unit/mg and 210 unit/mg, respectively. In TG1 transformat ArgRS was overproduced 120 times than that in TG1. The sequence of the cloned gene was different from the gene of E. coli JA200 at position 913 where C is changed to A corresponding Glu305 changed to Lys305 in ArgRS , but the activity of this enzyme was not affected by this substitution.