Soluble expression and characterization of disulfide bond-rich subdomains of membrane protein p185 in Escherichia coli.
- Author:
Liang-Wei LI
1
;
Hai-Bo LIU
;
Si-Yi HU
;
Dun LIANG
;
Lian-Sheng CHENG
;
Jing LIU
Author Information
1. School of Life Science, University of Science and Technology of China, Hefei 230027, China.
- Publication Type:Journal Article
- MeSH:
Antibodies, Monoclonal;
immunology;
Disulfides;
immunology;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Humans;
Receptor, ErbB-2;
biosynthesis;
genetics;
immunology;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
immunology;
Solubility;
Transfection
- From:
Chinese Journal of Biotechnology
2005;21(4):590-596
- CountryChina
- Language:Chinese
-
Abstract:
Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain I-II and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E. coli Origami B (DE3) pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10-15 mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain I-II and domain IV of p185 ECD and this method may also be used to express other disulfide bond rich proteins.
