Increased expression of the integral membrane proteins EGFR and FGFR3 in anti‐apoptotic Chinese hamster ovary cell lines

Abstract

Membrane proteins such as receptor tyrosine kinases (RTKs) have a vital role in many cellular functions, making them potential targets for therapeutic research. In this study, we investigated the coexpression of the anti-apoptosis gene Bcl-x(L) with model membrane proteins as a means of increasing membrane protein expression in mammalian cells. Chinese hamster ovary (CHO) cells expressing heterologous Bcl-x(L) and wild-type CHO cells were transfected with either epidermal growth factor receptor or fibroblast growth factor receptor 3. The CHO-Bcl-x(L) cell lines showed increased expression of both RTK proteins as compared with the wild-type CHO cell lines in transient expression analysis, as detected by Western blot and flow cytometry after 15 days of antibiotic selection in stable expression pools. Increased expression was also seen in clonal isolates from the CHO-Bcl-x(L) cell lines, whereas the clonal cell line expression was minimal in wild-type CHO cell lines. Our results demonstrate that application of the anti-apoptosis gene Bcl-x(L) can increase expression of RTK proteins in CHO cells. This approach may be applied to improve stable expression of other membrane proteins in the future using mammalian cell lines with Bcl-x(L) or perhaps other anti-apoptotic genes.

Comparative systeomics to elucidate physiological differences between CHO and SP2/0 cell lines

“…Membrane biogenesis is known to be enriched in murine cells, but these findings also suggest that this category of proteins may also be low in M-CHO cells 29 , 30 . Although M-CHO cells are widely used both for secreted and membrane protein expression, poor expression of membrane proteins has been previously reported 31 . In order to further examine the presence of key membrane and vesicle proteins in M-CHO cells, we applied three different enrichment methods to explore the M-CHO membranome.…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…Alternative approaches have been suggested to overcome toxicity issues associated with MP overexpression. Ohsfeldt et al (2012) designed an anti-apoptosis strategy involving coexpression of Bcl-xL gene (encodes for an antiapoptotic protein) aiming to prevent cell death by bioreactor stresses, nutrient depletion, toxin accumulation, and stresses due to folding and processing requirements for complex proteins such as MP. The authors observed that cell death are diminished due to the co-expression of the anti-apoptotic gene and transient production of two different receptors were improved (Ohsfeldt et al 2012).…”

Engineering cells to improve protein expression

“…Co-expression of the anti-apoptotic protein Bcl-x L in CHO cells improved the expression of epidermal growth factor receptor, fibroblast growth factor receptor 3 and receptor tyrosine kinases proteins [37]. Knock-out of the genes encoding the pro-apoptotic factors Bax and Bak in a CHO-K1 cell line improved cell viability, reduced levels of transfection-induced apoptosis and led to up to 4 fold higher antibody titers [38].…”