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在克隆含大肠杆菌亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因（ｌｅｕＳ）的３．２ｋｂＤＮＡ片段、并使ｌｅｕＳ在大肠杆菌ＴＧ１中高表达提高３５倍的基础上，将ｌｅｕＳ改造。分别将两种不同长度的ｌｅｕＳ１和ｌｅｕＳ２分别构建在表达载体ｐＫＫ－２３３－２和ｐＴｒｃ－９９Ｂ中，其中ｌｅｕＳ１比ｌｅｕＳ２多一段１３０ｂｐ的３′非翻译区。构建在表达载体ｐＴｒｃ－９９Ｂ中的基因片段ｌｅｕＳ２可将酶的表达量提高１３５倍，经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ和ＨＡ－Ｕｌｔｒａｇｅｌ二步柱层析纯化，得到ＳＤＳ－ＰＡＧＥ电泳一条带的酶。在重组过程中ｌｅｕＳ编码序列的第４位产生Ｃ→Ｇ突变，使ＬｅｕＲＳ第二位氨基酸由Ｇｌｎ突变为Ｇｌｕ，称变种酶为ＬｅｕＲＳ２Ｅ。ＬｅｕＲＳ２Ｅ的３种底物氨酰化反应中的动力学常数表明，２位氨基酸的变化对活力基本没有影响。
The leuS was engineered by cloning a 3.2 kb DNA fragment containing the LeuRS gene (leuS) of E. coli and increasing leuS by 35-fold over-expression in E. coli TG1. Two different lengths of leuS1 and leuS2 were constructed respectively in the expression vectors pKK-233-2 and pTrc-99B, in which leuS1 was a 130 bp 3 ’untranslated region more than leuS2. The leuS2 gene fragment, constructed in the expression vector pTrc-99B, increased the enzyme expression by 135-fold and was purified by two-step chromatography on a DEAE-Sepharose and HA-Ultragel gel to obtain a band of SDS-PAGE electrophoresis. In the process of recombination, the C → G mutation occurs at the 4th position of the leuS coding sequence, and the second amino acid of LeuRS is mutated from Gln to Glu, and the variant enzyme is LeuRS2E. The kinetic constants in the aminoacylation of the three substrates of LeuRS2E indicate that the change of amino acid 2 has no effect on the viability.