钩端螺旋体liprmA基因的表达及其产物的纯化与功能分析

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liprmA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。

Gene Expression, Purification and Functional Analysis of LiPrmA

Leptospira interrogans (L. interrogans) genomic DNA was used as template to amplify the full-length gene for ribosomal protein L11 methyltransferase (liPrmA) by PCR. The pET22b-/liprmA expression plasmid was successfully constructed in Escherichia 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. interrogans methylated under the presence of S-adenosyl-methionine (AdoMet).