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High‐level expression, purification and characterization of active human C1q and tumour necrosis factor‐related protein‐1 in E scherichia coli
Author(s) -
Li H.,
Hui X.,
Li K.,
Tang X.,
Hu X.,
Xu A.,
Wu D.
Publication year - 2014
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12280
Subject(s) - biomedicine , china , chinese academy of sciences , library science , human health , political science , biology , medicine , bioinformatics , computer science , environmental health , law
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, hCTRP 1 gene was expressed in Escherichia coli . In the E. coli expression system, a large amount of soluble thioredoxin (Trx)‐ hCTRP 1 fusion protein could be produced using the expression plasmid pET 32a (+) and induction with IPTG at 18°C, which accounts about 20% of the total soluble bacterial proteins. The recombinant Trx‐ hCTRP 1 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‐ hCTRP 1 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.