Cloning,expression and purification of recombinant human proinsulin C-peptide in E.coli
- VernacularTitle:重组人源胰岛素原C肽在大肠杆菌中的克隆、表达与纯化
- Author:
Xuejun WANG
;
Kai GU
;
Rongyue CAO
;
Jie LIN
;
Jie WU
;
Jingjing LIU
- Publication Type:Journal Article
- Keywords:
recombinant human proinsulin C-peptide;
gene fusion;
expression and purification;
asparaginase;
trypsin
- From:
Journal of China Pharmaceutical University
2006;(2):174-180
- CountryChina
- Language:Chinese
-
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.
