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Lett Appl Microbiol. 2014 Sep;59(3):334-41. doi: 10.1111/lam.12280. Epub 2014 Jun 14.

High-level expression, purification and characterization of active human C1q and tumour necrosis factor-related protein-1 in Escherichia coli.

Author information

1
Department of Life Sciences, The Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Huaihua College, Huaihua, China; The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

Abstract

C1q and tumour necrosis factor-related proteins (CTRPs) are a family of adiponectin paralogues. CTRP1 plays important biological functions in diabetes, obesity and hypertension. To further explore the physiological roles of human CTRP1 and its mechanisms of action, hCTRP1 gene was expressed in Escherichia coli. In the E. coli expression system, a large amount of soluble thioredoxin (Trx)-hCTRP1 fusion protein could be produced using the expression plasmid pET32a (+) and induction with IPTG at 18°C, which accounts about 20% of the total soluble bacterial proteins. The recombinant Trx-hCTRP1 fusion protein was purified to an approx. 95% purity using Ni-NTA affinity chromatography and Superdex G-75 column with a yield of about 28-mg protein from 1-l bacterial cultures. The purified recombinant Trx-hCTRP1 was shown to be active under in vivo and in vitro assay conditions.

SIGNIFICANCE AND IMPACT OF THE STUDY:

CTRP1 plays important biological functions and warrants further investigation. However, large-scale production of recombinant CTRP1 has been technically challenging, which becomes a major obstacle in the structural and functional analysis of this important family of proteins. To explore the possible clinical applications and mechanisms of its action, an efficient method to produce large amounts of active recombinant human CTRP1 is necessary. This study should facilitate basic functional and pharmacological studies of this important protein family.

KEYWORDS:

Escherichia coli; expression and purification; fusion protein; human C1q and tumour necrosis factor-related protein-1; pET32; thioredoxin

PMID:
24814641
DOI:
10.1111/lam.12280
[Indexed for MEDLINE]
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