title: Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins.
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.
authors.affiliation: College of Pharmacy, Dalian Medical University, Dalian, Liaoning Province, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Sun
authors.forename: Xue
authors.initials: X
authors.lastname: Feng
authors.forename: Jiao
authors.initials: J
authors.lastname: Qu
authors.forename: Wenqi
authors.initials: W
authors.lastname: Han
authors.forename: Zi
authors.initials: Z
authors.lastname: Liu
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.forename: Ming
authors.initials: M
authors.lastname: Zou
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Zhen
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D042361:Mammary Glands, Human / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial
chemical_list: D000906:Antibodies; D007544:Isopropyl Thiogalactoside; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C508053:ERBB2 protein, human