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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。
Using the genomic DNA of Leptospira as template, the full-length coding sequence of lip-rmA homologous to prmA in Leptospira was obtained by enzyme-linked polymerase chain reaction (PCR) and cloned into prokaryotic expression vector pET22b. By optimizing the conditions of E. coli culture and induction, the fusion protein containing the target protein reached a soluble expression level of 40 mg / L, accounting for about 40% of the total bacterial proteins. Purified by Ni-NTA His Bind affinity column to obtain the target protein with the purity higher than 95%. Amino acid sequence homology analysis showed that the liPrmA was highly consistent with the primary structure of the functional domain of the ribosomal protein L11 methylase in prokaryotes and eukaryotes. The activity analysis showed that the purified liPrmA had a L11 leptospiral protein The activity of a biotransferase.