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

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Abstract

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.

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Abbreviations

HER2:

Human epidermal growth factor receptor 2

EGFR:

Epidermal growth factor receptor

ECD:

Extracellular domain

IPTG:

Isopropyl-b-D-thiogalactopyranoside

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Acknowledgment

This work was financially supported by the Natural Science Foundation of Liaoning, China (2013023037). This article did not contain any studies with human subjects. All institutional and national guidelines for the care and use of laboratory animals were followed.

Conflict of Interest

The authors declared that there are no conflicts of interest.

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Authors and Affiliations

  1. College of Pharmacy, Dalian Medical University, Dalian, Liaoning Province, China

    Yong Sun, Jiao Qu, Wenqi Han, Zi Liu & Yuhong Zhen

  2. Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning Province, China

    Xue Feng, Xu Li & Ming Zou

  3. The Second Affiliated Hospital of Dalian Medical University, Dalian, Liaoning Province, China

    Jie Zhu

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  1. Yong Sun
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  2. Xue Feng
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Correspondence to Yuhong Zhen or Jie Zhu.

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Sun, Y., Feng, X., Qu, J. et al. Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins. Appl Biochem Biotechnol 176, 1029–1043 (2015). https://doi.org/10.1007/s12010-015-1627-x

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