Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins

• DOI: 10.1007/s12010-015-1627-x
• Publication Date: 2015-04-24T01:26:00Z
• Authors: Yong Sun; Xue Feng; Jiao Qu; Wenqi Han; Zi Liu; Xu Li; Ming Zou; Yuhong Zhen; Jie Zhu
• Keywords: Isopropyl Thiogalactoside; 0301 basic medicine; Mice, Inbred BALB C; 0303 health sciences; Receptor, ErbB-2; Blotting, Western; Genetic Vectors; Gene Expression; Antibodies; Recombinant Proteins; Protein Structure, Tertiary; 3. Good health; Mice; 03 medical and health sciences; Cell Line, Tumor; Escherichia coli; Animals; Humans; Electrophoresis, Polyacrylamide Gel; Female; Immunization; Transformation, Bacterial; Cloning, Molecular; Mammary Glands, Human

Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family. In this study, the whole extracellular domain gene of HER2 was amplified by RT-PCR from human breast cancer cell line SK-BR-3. The genes of membrane-distal region (A) and membrane proximal region (B) of HER2 extracellular domain were amplified from the cloned template, and then inserted into the expression vector pET-28a and pET-30a, respectively. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) cells and induced by isopropyl-b-D-thiogalactopyranoside (IPTG) for expression of proteins His-A and His-B. The expressed proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The optimization of culture conditions led us to accomplish the recombinant protein induction with 1.0 mM IPTG at 37 °C for 8 h, and both proteins were expressed in the insoluble form. Both proteins were purified under the denaturing condition using Ni-NTA sepharose column. Balb/c mice were immunized with the purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by ELISA testing and had good specificity by western blot detection. The HER2 ECD proteins His-A and His-B could be expressed in E. coli and were suitable for production of high titer antibodies against HER2 ECD.