Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker
- 7 Downloads
Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.
KeywordsChinese hamster ovary (CHO) cell Glutamine synthetase (GS) Simian vacuolating virus 40E (SV40E) promoter Stable transfection
This work was supported by Guangdong Provincial Science and Tech Project (2015A020211016); the Guangzhou Industry-Academia-Research Collaborative Innovation Project (201604016009).
Compliance with ethical standards
Conflict of interest
All the authors reviewed and agreed to submit this manuscript. The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
The study does not contain experiments using animals and human studies.
- 2.Deschenes I, Finkle CD, Winocour PD (1998) Effective use of BCH-2763, a new potent injectable direct thrombin inhibitor, in combination with tissue plasminogen activator (tPA) in a rat arterial thrombolysis model. Thromb Haemost 80(1):186–191Google Scholar
- 4.Chu L, Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 12(2):180–187Google Scholar
- 11.McKnight S, Tjian R (1986) Transcriptional selectivity of viral genes in mammalian cells. Cell 46(6):795–805Google Scholar
- 13.HHa WagnerR (1997) Mammalian cell biotechnology in protein production. Walter de GruyterGoogle Scholar
- 15.Girod PA, Nguyen DQ, Calabrese D, Puttini S, Grandjean M, Martinet D, Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods 4(9):747–753. https://doi.org/10.1038/nmeth1076 Google Scholar
- 16.Kim JD, Yoon Y, Hwang HY, Park JS, Yu S, Lee J, Baek K, Yoon J (2005) Efficient selection of stable chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon beta SAR element. Biotechnol Prog 21(3):933–937. https://doi.org/10.1021/bp049598v Google Scholar
- 17.Zhang F, Thornhill SI, Howe SJ, Ulaganathan M, Schambach A, Sinclair J, Kinnon C, Gaspar HB, Antoniou M, Thrasher AJ (2007) Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells. Blood 110(5):1448–1457. https://doi.org/10.1182/blood-2006-12-060814 Google Scholar
- 18.Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/technology 10(2):169–175Google Scholar
- 19.Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/technology 8(7):662–667Google Scholar
- 23.Byrne BJ, Davis MS, Yamaguchi J, Bergsma DJ, Subramanian KN (1983) Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat. Proc Natl Acad Sci USA 80(3):721–725Google Scholar
- 30.Zahn-Zabal M, Kobr M, Girod PA, Imhof M, Chatellard P, de Jesus M, Wurm F, Mermod N (2001) Development of stable cell lines for production or regulated expression using matrix attachment regions. J Biotechnol 87(1):29–42Google Scholar
- 33.Schimke RT (1984) Gene amplification in cultured animal cells. Cell 37(3):705–713Google Scholar