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Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

ABSTRACT: In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1? (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

SUBMITTER: Ebadat S

PROVIDER: S-EPMC5638317 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

Ebadat Saeedeh S Ahmadi Samira S Ahmadi Maryam M Nematpour Fatemeh F Barkhordari Farzaneh F Mahdian Reza R Davami Fatemeh F Mahboudi Fereidoun F

PloS one 20171012 10

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistron ...[more]

PMID: 29023479

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Project description:Abstract We previously demonstrated that increased monoclonal antibody productivity in dihydrofolate reductase (DHFR)-amplified CHO cells correlates with phosphorylated transcription factor-cytomegalovirus (CMV) promoter interactions. In this article, we extend the characterization to include CMV promoter methylation and its influence on NFκB and CREB1 transcription factor binding to the CMV promoter in two families of DHFR-amplified CHO cell lines. CMV promoter methylation was determined using bisulfite sequencing. To overcome Sanger-sequencing limitations due to high CG bias and multiple transgenes copies, pyrosequencing was used to determine the frequency of methylated cytosines in regions proximal to and containing the NFκB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor–DNA interactions. Antibodies to CREB1 and NFκB were used to immunoprecipitate formaldehyde-crosslinked protein-DNA fractions, followed by reverse transcription quantitative real-time polymerase chain reaction to quantitate the number of copies of CMV-promoter DNA bound to the various transcription factors. The relative unmethylated fraction at the CREB1 and NFκB consensus binding sites determined by pyrosequencing was correlated with transcription factor binding as determined by chromatin immunoprecipitation. Azacytidine treatment reduced methylation in all treated samples, though not at all methylation sites, while increasing transcription. Distinct promoter methylation patterns arise upon clonal selection in different families of cell lines. In both cell line families, increased methylation was observed upon amplification. In one family, the NFκB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NFκB consensus binding site was accompanied by more NFκB recruitment to the promoter region. Statement of Significance: In this work, we have characterized the methylation pattern in various positions along the cytomegalovirus (CMV) promoter driving the expression of the mAb heavy chain and light chain in different clones. The interplay between the methylated regions of transcription-factor consensus binding sites and the nuclear proteins influences the transcript levels, leading to higher productivity phenotypes. Our workflow demonstrates a way to identify influencers of high transcriptional rates and identifies opportunities for transcription factor screening and rational synthetic promoter design, toward enhancing specific productivity in commercially applicable manufacturing processes.

Dataset Information

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

Publications

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

Similar Datasets

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