Increased expression of the integral membrane protein ErbB2 in Chinese hamster ovary cells expressing the anti-apoptotic gene Bcl-xL.
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.
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Shannon
S
O'Connor
Edwin
E
Li
Brian S
BS
Majors
Lijuan
L
He
Jesse
J
Placone
Deniz
D
Baycin
Michael J
MJ
Betenbaugh
Kalina
K
Hristova
D000818:Animals; D017209:Apoptosis; D001761:Bleomycin; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics* / Q000378:metabolism
D001761:Bleomycin; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D051020:bcl-X Protein; C105427:Zeocin