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首页> 外文期刊>Preparative biochemistry & biotechnology: An international journal for rapid communication >New and Easy Strategy for Cloning,Expression,Purification,and Characterization of the 5S Subunit of Transcarboxylase from Propionibacterium f.shermanii
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New and Easy Strategy for Cloning,Expression,Purification,and Characterization of the 5S Subunit of Transcarboxylase from Propionibacterium f.shermanii

机译：克隆，表达，纯化和鉴定沙门氏丙酸杆菌转羧酶5S亚基的新简便策略

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摘要

Methylmalonyl CoA-oxalacetate transcarboxylase(EC 2.1.3.1)from Propionibacterium f.shermanii is a biotin dependent enzyme which transfers CO2 from methylmalonyl-CoA(MMCoA)to pyruvate via a carboxylated biotin group to form oxalacetate.It is composed of three subunits,the central cylindrical hexameric 12S subunit,the outer six dimeric 5S subunit,and the twelve 1.3S linkers.We here report the cloning,sequencing,expression,and purification of the 5S subunit.The gene was identified by matching the amino acid sequence with that of deposited in the NCBI database.For cloned 5S subunit sequence shows regions of high homology with that of pyruvate carboxylase and oxaloacetate decarboxylase.The gene encoding the 5S subunit was cloned into the pTXB1 vector.The expressed 5S subunit was purified to apparent homogeneity by a single step process by using Intein mediated protein ligation(IPL)method.The cloned 5S gene encodes a protein of 505 amino acids and of M_r 55,700.
机译：谢氏丙酸丙酸杆菌的丙二酸甲酯基CoA-草酰乙酸转羧酶（EC 2.1.3.1）是一种生物素依赖性酶，它通过羧化的生物素基团将CO2从甲基丙二酰-CoA（MMCoA）转移到丙酮酸中形成草酰乙酸。它由三个亚基组成中心圆柱形六聚体12S亚基，外部6个二聚5S亚基和12个1.3S接头。我们在此报告了5S亚基的克隆，测序，表达和纯化。克隆的5S亚基序列显示与丙酮酸羧化酶和草酰乙酸脱羧酶具有高度同源性的区域，将编码5S亚基的基因克隆到pTXB1载体中，表达的5S亚基通过一次纯化被纯化至表观同质性。通过使用Intein介导的蛋白质连接（IPL）方法进行分离过程。克隆的5S基因编码505个氨基酸的蛋白和M_r 55,700。

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