title: Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells.
abstract: Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains - I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using Western blot, mass spectrometry and circular dichroism (CD) spectroscopy.
authors.affiliation: Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71201, United States.
authors.forename: Shanthi
authors.initials: S
authors.lastname: Kanthala
authors.affiliation: Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849-5501, United States; St. Jude Children's Research Hospital, Memphis, TN 38105, United States.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Mill
authors.affiliation: Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849-5501, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Riese
authors.affiliation: Department of Biochemistry, University of Arkansas for Medical Sciences, United States.
authors.forename: Mihir
authors.initials: M
authors.lastname: Jaiswal
authors.affiliation: Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71201, United States. Electronic address: jois@ulm.edu.
authors.forename: Seetharama
authors.initials: S
authors.lastname: Jois
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004331:Drosophila melanogaster; D005260:Female; D006801:Humans; D010449:Peptide Mapping; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C508053:ERBB2 protein, human
keywords: Disulfide loops; Human epidermal growth factor receptor 2; Mass spectrometry; Protein secretion; Protein sequencing; S2 insect cell lines