八肋游仆虫赖氨酰tRNA合成酶的表达纯化及活性分析

来源 :中国生物化学与分子生物学会第十二届会员代表大会暨2018年全国学术会议 | 被引量 : 0次 | 上传用户:leon2000
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  赖氨酰-tRNA合成酶(Lysyl-tRNA synthetase,LysRS)是蛋白质生物合成中的关键酶,能够催化赖氨酸和相应tRNA的氨酰化反应.除氨酰化作用外,LysRS还参与了转录调控、信号传导及免疫应答等多种生命活动.本研究对单细胞原生动物八肋游仆虫(Euplotes octocarinatus)的LysRS进行了研究,所得结果如下:生物信息学分析表明,八肋游仆虫具有两个LysRS基因(EoLysRS1和EoLysRS2),二者的氨基酸序列一致性为39.8%,其中EoLysRS2含有线粒体导肽,推测其定位于线粒体,且在翻译过程中需发生一次+2位移码,而EoLysRS1不含线粒体导肽,推测其定位于细胞质;将EoLysRS1基因克隆后构建重组表达质粒pET28a-LysRS1,在大肠杆菌Transetta(DE3)中获得高效表达,经镍柱亲和层析后获得纯度较高的重组蛋白EoLysRS1,表达产物经Western blotting检测为阳性;对EoLysRS1的酶活进行测定,结果表明大肠杆菌表达的EoLysRS1具有活性.
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