Expression, purification and activity characterization of a thioredoxin peroxidase from Branchiostoma belcheri Tsingtaunese
10.3724/SP.J.1008.2008.00781
- Author:
Jian LIAO
1
Author Information
1. Institute of Clinical Laboratory Medicine
- Publication Type:Journal Article
- Keywords:
Branchiostoma belcheri Tsingtaunese;
Enzymatic activity;
Prokaryotic expression;
Protein purification;
Structure;
Thioredoxin peroxidase
- From:
Academic Journal of Second Military Medical University
2010;29(7):781-786
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct a prokaryotic expression vector carrying thioredoxin peroxidase (TPx) gene of Branchiostoma belcheri Tsingtaunese and express it in E. coli. Methods: The cDNA fragments encoding TPx were obtained from Branchiostoma belcheri Tsingtaunese and were cloned into the expression vector pET-32a (+) M; the product was used to transform BL21 (DE3) cells and expression of TPx protein was induced by IPTG. The recombinant TPx was expressed as a histidine fusion protein in E. coli and was purified with Ni chromatography and SP cation exchange chromatography. The expression and purification of TPx were analyzed by SDS-PAGE; the activity and structure of the protein were analyzed. Results: The recombinant plasrnid pET-32a (+) M-TPx was highly expressed in E. coli. The purity of the protein was over 90% after purification; the molecular weight of the protein monomer was 24 460. The recombinant TPx protein existed as a mixture of both dimer and monomer. The recombinant TPx had a significant thiol-dependent peroxidase activity in the presence of dithiothreitol (DTT) and it could protect plasmid DNA and thiol-protein from damages caused by metal catalyzed oxidation (MCO). Conclusion: The recombinant TPx protein has been successfully expressed in E. coli; its activity is closely related to the advanced structures.
