钩端螺旋体liprmA基因的表达及其产物的纯化与功能分析  

Gene Expression, Purification and Functional Analysis of liprmA

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作  者:孙艳菊[1] 周忠卫[2] 姚建秀[2] 孙体昌[1] 许琰艳[2] 文雅[2] 鲍时来[2] 

机构地区:[1]北京科技大学土木与环境工程学院,北京100083 [2]中国科学院遗传与发育生物学研究所,北京100101

出  处:《遗传》2005年第5期792-796,共5页Hereditas(Beijing)

摘  要:以钩端螺旋体基因组DNA为模板,通过酶联聚合反应(PCR)得到钩端螺旋体中prmA的同源基因lip-rmA的全基因编码序列,并克隆到原核表达载体pET22b中。通过优化大肠杆菌培养和诱导条件,含目的蛋白的融合蛋白可溶表达量达到40 mg/L,约占菌体总蛋白的40%。经Ni-NTA His Bind亲和柱纯化,得到纯度大于95%的目的蛋白。氨基酸序列同源性分析显示liPrmA与原核生物和真核生物的核糖体蛋白L11甲基化转移酶的功能域一级结构高度一致;活性分析显示,纯化的liPrmA有钩端螺旋体核糖体蛋白L11甲基化转移酶的活性。Leptospire interrogens (L. interrogens) genomic DNA was used as template to amplify the full-length gene for ribosomal protein L11 methyltransferase (liPrmA) by POR. The pET22b-/liprrnA expression plasmid was successfully constructed in Escherichie coli (E. coli. ) strain TOP10 and confirmed by restriction enzyme digest and sequencing. Through optimizing expression of the recombinant liPrmA-6xHis fusion protein in expression host E. coli. BL21, the yield of soluble target protein reached 40 mg (liter culture)^-1. The LiPrmA was purified to apparent homogeneity in a single step using Ni-NTA His Bind chromatography. Amino acid homologous analysis showed that liPrmA shared significant identity with other prokaryotic PrmA and eukaryotic putative PrmA in the catalytic region including AdoMet binding domain. Methylation activity experiments showed purified liPrmA was able to catalyze the ribosomal protein L11 of L. interrogens methylated under the presence of S-adenosyl-methionine (AdoMet).

关 键 词:钩端螺旋体甲基化转移酶 钩端螺旋体 基因表达 纯化 大肠杆菌 

分 类 号:R377[医药卫生—病原生物学]

 

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