Cloning,expression and purification of recombinant human proinsulin C-peptide in E.coli / 中国药科大学学报
Journal of China Pharmaceutical University
; (6): 174-180, 2006.
Article
in Chinese
| WPRIM (Western Pacific)
| ID: wpr-434064
Responsible library:
WPRO
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.
Full text:
Available
Database:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Journal of China Pharmaceutical University
Year:
2006
Document type:
Article