Purification and characterization of recombinant extracellular domain of human HER2 from Escherichia coli.
The human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family, and it plays an important role in the development of many human adenocarcinomas. The extracellular domain (ECD) of HER2 is an ideal target for therapeutic approaches. In order to obtain large quantities of active HER2 ECD protein for biochemical and structural analysis and for detecting anti-HER2 ECD antibodies in serum, a systematic assessment of optimal parameters for the refolding of the glutathione S-transferase (GST) fusion protein was carried out. After the GST-HER2 ECD inclusion bodies were solubilized with denaturation buffer containing 8M urea, an approach was then used to optimize refolding parameters. This approach utilized dilution of denatured and reduced GST-HER2 ECD into different refolding buffers using orthogonal design method. Optimal refolding was obtained in an alkaline buffer containing reduced and oxidized glutathione, and subsequent incubation at 4 degrees C for 24h. After purification with glutathione Sepharose 4B and PreScission protease cleavage of the fusion protein, 8.9mg of recombinant HER2 ECD was obtained from 1L of Escherichia coli. Rabbit polyclonal antibodies against HER2 ECD were obtained. The purified protein was found to be immunogenic and useful for immunodiagnostic studies of serum HER2 ECD and its antibodies by using enzyme-linked immunosorbent assay (ELISA).
Liu
Xiaorong
X
Cancer Research Institute, Central South University, Changsha, China.
He
Zhimin
Z
Zhou
Min
M
Yang
Fang
F
Lv
Hui
H
Yu
Yanhui
Y
Chen
Zhuchu
Z
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000097:blood; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D017931:DNA Primers; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins