[Soluble expression and characterization of disulfide bond-rich subdomains of membrane protein p185 in Escherichia coli].
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.
Li
Liang-Wei
LW
School of Life Science, University of Science and Technology of China, Hefei 230027, China.
Liu
Hai-Bo
HB
Hu
Si-Yi
SY
Liang
Dun
D
Cheng
Lian-Sheng
LS
Liu
Jing
J
D000911:Antibodies, Monoclonal / Q000276:immunology; D004220:Disulfides / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012995:Solubility; D014162:Transfection
D000911:Antibodies, Monoclonal; D004220:Disulfides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins