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首页> 外文期刊>The protein journal >Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase
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Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase

机译：异源大肠杆菌的表达，纯化和表征的GrmA氨基糖苷抗性甲基转移酶。

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摘要

The mechanism of resistance to aminoglyco-sides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resis-tance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His_6-EK-GrmA and His_6-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of ami-noglycoside-resistance enzymes.
机译：直到最近，仅在产生抗生素的微生物中描述了基于其靶标16S rRNA甲基化对氨基糖苷侧产生抗性的机制。但是，现在也已在众多临床革兰氏阴性致病菌中鉴定出了等效的甲基转移酶。我们已经从庆大霉素复合物的生产商Micromonospora purpurea克隆，表达和纯化了GrmA，一种氨基糖苷抗性甲基转移酶。创建了两个表达具有N端6x组氨酸标签的蛋白的载体，所述蛋白具有和不具有肠激酶识别，分别产生蛋白His_6-EK-GrmA和His_6-GrmA。在体内证明了两种重组蛋白的活性。经过优化的表达和天然纯化后，两种蛋白变体在体外甲基化测定中均具有活性。这项工作为重要的氨基糖苷抗性酶的这一成员奠定了基础，为将来进行详细的生化，结构和药理研究奠定了基础。

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