Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells

https://doi.org/10.1016/j.pep.2015.09.001Get rights and content

Highlights

  • HER2 protein extracellular domain and domain IV were expressed in S2 insect cells.

  • Proteins were secreted into the medium in fully folded form.

  • Proteins were purified and stability was analyzed by circular dichroism spectroscopy.

  • Sequence analysis was done by tandem mass spectrometry.

  • Sequence information was obtained from collision induced dissociation fragmentation.

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.

Introduction

Human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) is involved in regulating cell growth, proliferation and survival. It is overexpressed in about 30% of human breast cancers and this expression is correlated with aggressive tumor phenotypes. This overexpression results in constitutive HER2 dimerization and signaling, typified by phosphorylation of HER2 cytoplasmic tyrosine residues [1], [2], [3], [4]. Major clinical milestones in the treatment of cancers that overexpress HER2 are the FDA approval of trastuzumab and pertuzumab, humanized monoclonal antibodies that target HER2 [5], [6], [7]. Therefore, there is significant interest in the mechanisms of HER2 signal transduction and in strategies for disrupting HER2 signaling.

HER2 is a canonical receptor tyrosine kinase with several distinct functional motifs. HER2 consists of an extensive (∼600 amino acid residues) extracellular domain; a hydrophobic, single pass transmembrane domain; a cytoplasmic tyrosine kinase domain; and a series of cytoplasmic tyrosine residues that serve as sites of phosphorylation. The HER2 extracellular domain (ECD) can be divided further into four functional domains (I, II, III, and IV) [8]. Domain IV (DIV) is near the transmembrane domain and stabilizes the protein–protein interaction between HER2 and its dimerization partner. Mutations in DIV impair phosphorylation [9] and signaling by heterodimers containing HER2. Nonetheless, the information regarding the role of the C-terminal region of DIV in heterodimerization is controversial [10]. Consequently, we have designed peptidomimetics that can bind to domain IV of HER2 and modulate HER2 signaling [11], [12], [13], [14], [15], [16]. These peptidomimetics inhibit the protein–protein interaction of EGFR:HER2 and HER2:HER3 heterodimers [14]. In order to investigate the binding mode of such molecules on domain IV of HER2, structural elucidation of the HER2 extracellular domain–peptidomimetic complex is important.

Previous findings suggest that the ECD of EGFR family members can be expressed successfully in a number of host systems [17], [18], [19], [20], [21], [22]. The aim of this project is to express and purify the HER2 ECD to study its binding to various antagonists. We used recombinant S2 (Schneider2) cells for this purpose. S2 cells are derived from primary cultures of late stage embryonic cells of Drosophila melanogaster [23], [24]. The rationale for using S2 cells for obtaining our protein is that these cells are easy to culture and grow as a semi-adherent monolayer at room temperature without CO2. Moreover, S2 cells can be directed to secrete recombinant proteins into the culture medium in their native form, making for easy recovery and purification without using denaturing conditions. In contrast, recombinant proteins expressed in Escherichia coli are typically recovered following lysis of the E. coli under denaturing conditions, which necessitates refolding of the recombinant proteins following recovery. In addition, unlike recombinant proteins expressed in E. coli, recombinant proteins expressed in S2 cells will form intramolecular disulfide bonds. This is particularly important for the HER2 DIV, which contains intramolecular disulfide bridges and is structurally divided into seven modules [25], [26], [27] (Fig. 1) that are difficult to reconstitute from a recombinant protein expressed by E. coli. As a result of these advantages, S2 cells are being used for a variety of purposes, including the production of vaccine antigens and other heterologous proteins. It is noteworthy that the S2 system may allow for higher yields of these proteins than bacterial and yeast expression systems [28], [29].

Section snippets

Reagents

Drosophila Schneider2 (S2) cells, the Drosophila expression system-inducible/secreted kit, calcium phosphate transfection kit, hygromycin, fungizone, l-glutamine, probond nickel-chelating resin and purification columns were purchased from Life Technologies (Grand Island, NY). The Amicon 8400-stirred ultrafiltration apparatus was obtained from Millipore Corp. (Bellerica, MA). Fetal bovine serum (FBS) was obtained from ATCC (Manassas, VA). Schneider insect cell medium powder and copper(II)

Results and discussion

Transfection of the expression vectors was confirmed by fluorescence microscopy of the GFP transfected control cells (Fig. 2). The standard temperature for S2 cells is 27–28 °C [31]. However, at this temperature the S2 cells transfected with HER2 expression vector grew slowly. Therefore, we determined experimentally that 22 °C is optimal for cell growth (data not shown). Enhanced production of proteins using non-standard cell culture conditions, including propagation at 22 °C has been reported in

Conclusion

In conclusion, the HER2 ECD and DIV proteins were successfully expressed and purified in S2 insect cells. Since the method of purification does not involve protein-denaturing conditions, the secondary structure of the proteins remains in the native form and is stable at room temperature >30 h. Hence an insect cell based method for expression and purification of HER2 ECD and DIV proteins has been developed in this project.

Authors’ contributions

Shanthi P. Kanthala: Carried out transfection, expression, purification, Western blot, and circular dichroism experiments; contributed to writing.

Christopher Mill: Constructed the vector for HER2 ECD domain, transfected the S2 cells with the vector, and contributed to writing.

David Riese: Directed the construction of the HER2 ECD vector in his lab, provided the transfected S2 cells to Dr. Jois, and contributed to writing.

Mihir Jaiswal: Carried out the analysis of the mass spectrometry data to

Acknowledgements

The authors would like to thank Dr. Jeonghoon Lee, mass spectrometry facility at Louisiana State University for helping with analysis of the proteins in this work. Authors would also like to thank UAMS proteomics core and Dr. Boris Zybaylov at the University of Arkansas for Medical Sciences for helping with the analysis of mass spectral data of proteins. The project was funded from the National Institute of General Medical Sciences of the National Institute of Health-United States under Grant

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