Soluble expression and purification of human alpha-defensin-5 in Escherichia coli.
- Author:
Aiping WANG
1
;
Yongping SU
;
Tianmin CHENG
;
Zhongmin ZOU
;
Junping WANG
Author Information
1. Institute of Combined Injury of the People's Liberation of Army, National Key Laboratory of Trauma, Burn and Combined Injury, Third Military Medical University, Chonqqing 400038, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Humans;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
isolation & purification;
Solubility;
Transformation, Bacterial;
alpha-Defensins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2008;24(2):291-296
- CountryChina
- Language:Chinese
-
Abstract:
DNA fragment containing human alpha-defensin 5 mature peptide (mHD-5) coding sequence with biased codons of E. coli was amplified by PCR, which was subsequently cloned into the plasmid pMAL-p2x in order to create pMAL-p2x-mHD-5 expression vector. The plasmid pMAL-p2x-mHD-5 was transferred into engineered strain BL21(DE3) to express heterogeneous fusion protein (MBP-mHD-5). The soluble MBP-mHD-5 targeted protein inducible expressed by IPTG was accounted for about 30% under optimized conditions. The recombinant mHD-5 (rmHD-5) peptide was successfully purified through a separation process including affinity chromatography, Factor Xa digestion and ion exchange chromatography. The bioactivity of rmHD-5 was examined by bacteria-inhibition tests in liquid culture. The growth of E. coli ATCC25922 was dramatically suppressed with an inhibition rate of 90%, with the presence of 62.5 microg/mL rmHD-5 in the media. These results indicate that the strategy of soluble expression of fusion protein in E. coli can be a useful and practical way to produce bioactive defensins.
