Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker
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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.
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