重组人源胰岛素原C肽在大肠杆菌中的克隆、表达与纯化(英文)  

Cloning ,expression and purification of recombinant human proinsulin C-peptide in E.coli

作　　者：王学军[1] 顾凯[1] 曹荣月[1] 林洁[1] 吴洁[1] 刘景晶[1] 

机构地区：[1]中国药科大学生命科学与技术学院微基因药物实验室,南京210009

出　　处：《中国药科大学学报》2006年第2期174-180,共7页Journal of China Pharmaceutical University

基　　金：theNationalHighTechnologyResearch&DevelopmentProgramofChina(863Program)(No.2005AA217032);NationalNaturalScienceFoundationofChina(No.39870175)andNaturalSci-enceFoundationofJiangsuProvince(No.BG2001011)

摘　　要：目的:构建一种新型表达载体(pEDCC),表达并纯化得到人源胰岛素原C肽。方法:将编码截短的门冬酰胺酶突变体(ansB-C),天然C肽,人IgG1铰链区(hinge),额外的酸敏感二肽(DP)以及富含碱性氨基酸的8肽(KRKRKKSR)的核苷酸序列依次分别插入pET28a载体中,构建表达载体pEDCC。在乳糖的诱导下,融合蛋白ansB-C-hinge-DPKRKRKKSRNGS-GR-C-peptide以包涵体形式高效表达。融合蛋白经洗涤和乙醇分级沉淀纯化后,通过酸水解将PKRKRKKSRNGSGR-C-peptide释放出来。C肽N端14肽用胰蛋白酶切割,通过DE52柱与C-peptide分离。结果:构建的表达载体pEDCC序列正确,融合蛋白经分离纯化得到了高纯度的重组人源胰岛素原C肽。结论:以截短的门冬酰胺酶作为融合伙伴,并以富含碱性氨基酸的8肽调节等电点是一种生产重组人源胰岛素原C肽的有效方法。Aim： To obtain recombinant human proinsulin C-peptide, a novel expression vector pEDCC was constructed to facilitate the expression and purification of C-peptide. Methods： Gene fragments encoding a truncated asparaginase fragment mutant,native C-peptide,a hinge fragment of human IgG1, an extra acid-labile dipeptide and a basic-amino- acid-riched octopeptide were introduced in turn into plasmid pET28a. The fusion protein ansB-C-hinge- DPKRKRKKSRNGSGR-C-peptide was expressed effectively as inclusion bodies after induced by lactose and partially purified by means of washing and ethanol fractionation. After being hydrolyzed, the polypepfide PKRKRKKSRNGSGR- C-peptide was liberated from the fusion partner. The N-terminal tetradecapeptide extension of C-peptide was subsequently cleaved by trypsin and removed by DE52 column. Results. The nucleotides sequence of plasmid pEDCC was confirmed to be identical with that of designed fusion protein. Recombinant human proinsulin C-peptide was obtained with high purity after purification. Conclusion：Employing truncated asparaginase as the fusion partner and basic-amino-acid-riched octopeptide to modulate isoelectric point is an effective approach to produce recombinant human proinsulin C-peptide.

关 键 词：重组人源胰岛素原C肽 基因融合 表达与纯化 门冬酰胺酶 胰蛋白酶 

分 类 号：R91[医药卫生—药学]