利用大肠杆菌甲硫氨酸氨基肽酶去除重组人干扰素-α2b N端的起始甲硫氨酸  

Processing of the N-terminal initiation methionine from recombinant human interferon-α2b by Escherichia coli methionine aminopeptidase

作　　者：王中安[1] 邹文艺[2] 刘磊[3] 范清林[2] 宋礼华[1,2] 

机构地区：[1]安徽医科大学生物化学与分子生物学教研室,合肥230032 [2]安徽安科生物工程(集团)股份有限公司,合肥230088 [3]安徽大学生命科学学院,合肥230039

出　　处：《安徽医科大学学报》2012年第3期229-233,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家"重大新药创制"科技重大专项(编号:2008ZX09203-003)

摘　　要：目的构建重组大肠杆菌甲硫氨酸氨基肽酶(re-MAP)的原核表达体系,并利用表达的reMAP切除重组人干扰素-α2b(rhIFN-α2b)N末端的初始甲硫氨酸。方法将PCR扩增得到的大肠杆菌MAP基因克隆到表达载体质粒pACYCDuet-1中,并在宿主菌E.coli BL21中通过IPTG诱导进行可溶性表达,经SDS-PAGE和Western blot分析及活性检测后,通过两种方法作用rhIFN-α2b:①将纯化后reMAP和rhIFN-α2b在体外适当的缓冲液中作用;②构建reMAP和rhIFN-α2b的共表达体系,实现在细胞内对rhIFN-α2b进行作用,并对两种作用方式的结果进行比较。结果 reMAP和rhIFN-α2b均得到正确表达,酶活性检测结果表明reMAP具有水解N端甲硫氨酸的活性,两种方法作用后的rhIFN-α2b经氨基酸序列测定后显示N端起始甲硫氨酸均被切除,其中体内作用切除率>94%、体外作用切除率>96%。结论 reMAP可以用于rhIFN-α2b N末端的初始甲硫氨酸的切除。Objective To construct the prokaryotic expression system of recombinant Escherichia coli（E.coil） methionine aminopeptidase（reMAP） and use the reMAP to remove the N-terminal initiator methionine residue from recombinant human interferon-α2b（rhIFN-α2b）.Methods The sequences of E.coli map gene was amplified with PCR and then cloned into the IPTG-controlled expression vector pACYCDuet-1,transformed into competent cell of E.coli BL21.The positive recombinant clones were expressed under IPTG induction,and the expression productions were investigated by SDS-PAGE,Western blot analysis and activity assay.Then determined the processing of rhIFN-α2b by purified MAP in vitro and coexpression of MAP and rhIFN-α2b in vivo.Results The analysis of SDS-PAGE and Western blot indicated that both MAP and rhIFN-α2b were expressed correctly.Activity assay indicated that the reMAP had the ability to remove the N-terminal methionine from polypeptides.The N-terminal sequencing of rhIFN-α2b indicated that the N-terminal initiator Met removal rate of processing was above 94% in vivo,while which processing was above 96% in vitro.Conclusion The reMAP can be used to remove the N-terminal initiator methionine residue from rhIFN-α2b.

关 键 词：甲硫氨酸氨基肽酶 干扰素-Α2B N端甲硫氨酸 基因表达 大肠杆菌 

分 类 号：Q784[生物学—分子生物学] Q815