中国药科大学学报2006，Vol.37Issue(2)：174-180,7.
重组人源胰岛素原C肽在大肠杆菌中的克隆、表达与纯化
Cloning,expression and purification of recombinant human proinsulin C-peptide in E.coli
摘要
Abstract
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 polypeptide 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Key words
recombinant human proinsulin C-peptide/gene fusion/expression and purification/asparaginase/trypsin分类
医药卫生引用本文复制引用
王学军,顾凯,曹荣月,林洁,吴洁,刘景晶..重组人源胰岛素原C肽在大肠杆菌中的克隆、表达与纯化[J].中国药科大学学报,2006,37(2):174-180,7.基金项目
This project was supported by the National High Technology Research & Development Program of China (863 Program) (No.2005AA217032) （863 Program）
National Natural Science Foundation of China (No.39870175)and Natural Science Foundation of Jiangsu Province (No.BG2001011). （No.39870175）
