Increased expression of the integral membrane protein ErbB2 in Chinese hamster ovary cells expressing the anti-apoptotic gene Bcl-xL

Abstract

Receptor tyrosine kinases (RTKs) are the second largest family of membrane receptors and play a key role in the regulation of vital cellular processes, such as control of cell growth, differentiation, metabolism, and migration. The production of whole-length RTKs in large quantities for biophysical or structural characterization, however, is a challenge. In this study, a cell engineering strategy using the anti-apoptotic Bcl-2 family protein, Bcl-x L , was tested as a potential method for increasing stable expression levels of a recombinant RTK membrane protein in Chinese hamster ovary (CHO) cells. Wild type and CHO cells stably overexpressing heterologous Bcl-x L were transformed with the gene for a model RTK membrane protein, ErbB2, on a plasmid also containing the Zeocin resistance gene. While CHO cells exhibited a gradual decrease in expression with passaging, CHO-bcl-x L cells offered an increased and sustained level of ErbB2 expression following continuous passaging over more than 33 days in culture. The increased ErbB2 expression in CHO-bcl-x L cells was evident both in stable transfected pools and in clonal isolates, and demonstrated both in western blot analysis and flow cytometry. Furthermore, the sustained high-level protein expression in CHO-bcl-x L cells does not alter the correct membrane localization of the ErbB2 protein. Our results demonstrate that cellular engineering, specifically anti-apoptosis engineering, can provide increased and stable ErbB2 membrane protein expression in mammalian cells. This approach may also be useful for other membrane proteins in which large quantities are needed for biophysical and structural studies.

A review of the mammalian unfolded protein response

“…Typical mammalian production hosts for this class of product include immortalized Chinese Hamster Ovary (CHO) cells (Wurm, 2004) or other mammalian cell types, such as murine lymphoid cells (NS0, SP2/0) (Khoo et al, 2007). These cell lines are often engineered or selected for high specific protein production rates (Seth et al, 2007, Barnes and Dickson, 2006), high growth rates (Khoo et al, 2007) and reduced apoptosis (Connor et al, 2009). However, beyond the robustness of the production host, protein production may also be sensitive to intracellular processing bottlenecks affecting transcription, translation or post-translational modifications.…”

Receptor tyrosine kinase transmembrane domains

“…One of the major experimental challenges in these studies is the expression of fulllength RTKs in sufficient quantities for biophysical and structural characterization. While there have been some important advances in RTK overexpression, such as preparation of large quantities of pure EGFR, 10 and long-term Neu/ErbB2 expression in engineered cells expressing anti-apoptotic proteins, 11 biophysical and structural characterization of interactions is carried out predominantly with the isolated domains, either extracellular, catalytic or TM. Thus, knowledge about the synergy between these RTK domains in signaling is lacking, and it is not yet clear how exactly ligand binding and structural changes in the extracellular domains are coupled to phosphorylation in the catalytic domains.…”

Physical–chemical principles underlying RTK activation, and their implications for human disease

“…High resolution structures of full-length RTKs are not available yet, due to experimental challenges in the expression of full-length RTKs in large quantities (117). Since the determination of full-length RTK dimer structures is challenging, studies of the effect of pathogenic mutations on dimerization, ligand binding, and phosphorylation may help us deduce possible structural changes in mutant RTK dimers.…”