Related Experiment Video
Updated: Apr 4, 2026
Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes
Published on: September 29, 2023
Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells
Shanthi Kanthala1, Christopher P Mill2, David J Riese3
1Basic Pharmaceutical Sciences, School of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71201, United States.
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.
Insights
Researchers produced pure, folded HER2 extracellular domain (ECD) and domain IV (DIV) proteins. This is crucial for studying how peptidomimetics bind to HER2, aiding breast cancer research.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Overexpression of human epidermal growth factor receptor 2 (HER2) is linked to aggressive breast cancer.
- The precise role of HER2's extracellular domain IV (DIV) in receptor function remains unclear.
- Previous work identified peptidomimetics that inhibit HER2:HER3 heterodimerization.
Purpose of the Study:
- To express and purify recombinant HER2 extracellular domain (ECD) and domain IV (DIV) proteins.
- To obtain properly folded, pure HER2 proteins for detailed binding interaction studies.
- To facilitate research into peptidomimetic inhibition of HER2 signaling.
Main Methods:
- Utilized the Drosophila melanogaster Schneider2 (S2) cell line and the Drosophila expression system (DES).
- Expressed and purified secreted HER2 ECD and DIV proteins from conditioned medium.
- Employed filtration, ultrafiltration, dialysis, and nickel affinity chromatography for purification.
- Analyzed purified proteins using Western blot, mass spectrometry, and circular dichroism (CD) spectroscopy.
Main Results:
- Successfully expressed and purified recombinant HER2 ECD and DIV proteins.
- Confirmed the purity and proper folding of the recombinant proteins using analytical techniques.
- Established a reliable method for producing functional HER2 protein fragments.
Conclusions:
- The study provides essential, pure recombinant HER2 ECD and DIV proteins.
- These purified proteins are suitable for detailed biophysical and biochemical analyses.
- This work supports further investigation into HER2-targeted therapies for breast cancer.
