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
de Zh
| WPRIM
| ID: wpr-305197
Bibliothèque responsable:
WPRO
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.
Texte intégral:
1
Indice:
WPRIM
Sujet Principal:
Solubilité
/
Protéines de fusion recombinantes
/
Transfection
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Récepteur ErbB-2
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Disulfures
/
Allergie et immunologie
/
Escherichia coli
/
Vecteurs génétiques
/
Génétique
/
Métabolisme
Type d'étude:
Prognostic_studies
Limites du sujet:
Humans
langue:
Zh
Texte intégral:
Chinese Journal of Biotechnology
Année:
2005
Type:
Article