Soluble expression and characterization of disulfide bond-rich subdomains of membrane protein p185 in Escherichia coli / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 590-596, 2005.
Article
in Chinese
| WPRIM
| ID: wpr-305197
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Solubility
/
Recombinant Fusion Proteins
/
Transfection
/
Receptor, ErbB-2
/
Disulfides
/
Allergy and Immunology
/
Escherichia coli
/
Genetic Vectors
/
Genetics
/
Metabolism
Type of study:
Prognostic study
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2005
Type:
Article
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