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采用PCR技术从草分枝杆菌(Mycobacterium phlei)基因组中扩增出亮氨酰-t RNA合成酶基因leu RS,依次克隆入p MD19-T Simple克隆载体及p ET28a(+)表达载体,并在大肠埃希菌(Escherichia coli)BL21(DE3)中表达。表达产物用Ni~(2+)螯合的His Trap~(TM) HP亲和色谱纯化,测定纯化所得目的蛋白的酶活力。结果显示,经PCR扩增得到2.8 kb的DNA片段,重组质粒p ET28a(+)-leu RS经酶切鉴定和测序分析,表明构建正确。SDS-PAGE显示,有与目的蛋白大小相当(相对分子质量约1.10×10~5)的蛋白质表达,表达的重组蛋白占菌体可溶性蛋白的32.8%,纯化后的蛋白质纯度达93.8%,酶活力为13.2 u/ml。
The leucyl-t RNA synthase gene leu RS was amplified by PCR from the Mycobacterium phlei genome and cloned into pMD19-T Simple cloning vector and pET28a (+) expression vector in turn, Escherichia coli BL21 (DE3). The expressed product was purified by Ni ~ (2+) chelated His Trap ~ (TM) HP affinity chromatography and the activity of the purified protein was determined. The results showed that the 2.8 kb DNA fragment was amplified by PCR. The recombinant plasmid p ET28a (+) - leu RS was identified by restriction analysis and sequencing analysis, indicating that it was correctly constructed. SDS-PAGE showed that the protein of the same size as the target protein (molecular weight of about 1.10 × 10 ~ 5) expressed 32.8% of recombinant soluble protein, purified protein purity of 93.8%, the enzyme activity 13.2 u / ml.