Article

Recombinant protein expression by targeting pre-selected chromosomal loci

Helmholtz Centre for Infection Research, Braunschweig, Germany.
BMC Biotechnology (Impact Factor: 2.59). 12/2009; 9:100. DOI: 10.1186/1472-6750-9-100
Source: PubMed

ABSTRACT Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.
We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.
RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.

Download full-text

Full-text

Available from: Dagmar Wirth, Jul 01, 2015
0 Followers
 · 
175 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the years Chinese hamster ovary (CHO-) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest and have thereby left the identification of bottlenecks a challenging task. For comparison of different producer cell lines derived from various transfections a system is highly needed that provides control over transgene expression behavior. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase mediated cassette exchange (RMCE) using the FLP/FRT system. The RMCE competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F-) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single chain Fc-fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and mRNA levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes
    Applied Microbiology and Biotechnology 08/2014; · 3.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Over the years Chinese hamster ovary (CHO-) cells have emerged as the major host for expressing biotherapeutic proteins. Traditional methods to generate high-producer cell lines rely on random integration(s) of the gene of interest and have thereby left the identification of bottlenecks a challenging task. For comparison of different producer cell lines derived from various transfections a system is highly needed that provides control over transgene expression behavior. This motivated us to develop a novel "DUKX-B11 F3/F" cell line to target different single-chain antibody fragments into the same chromosomal target site by recombinase mediated cassette exchange (RMCE) using the FLP/FRT system. The RMCE competent cell line contains a gfp reporter fused to a positive/negative selection system flanked by heterospecific FRT (F-) variants under control of an external CMV promoter, constructed as "promoter trap". The expression stability and FLP accessibility of the tagged locus was demonstrated by successive rounds of RMCE. As a proof of concept, we performed RMCE using cassettes encoding two different anti-HIV single chain Fc-fragments, 3D6scFv-Fc and 2F5scFv-Fc. Both targeted integrations yielded homogenous cell populations with comparable intracellular product contents and mRNA levels but product related differences in specific productivities. These studies confirm the potential of the newly available "DUKX-B11 F3/F" cell line to guide different transgenes into identical transcriptional control regions by RMCE and thereby generate clones with comparable amounts of transgene mRNA. This new host is a prerequisite for cell biology studies of independent transfections and transgenes.
    Applied Microbiology and Biotechnology 08/2014; DOI:10.1007/s00253-014-6011-1 · 3.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.
    Metabolic Engineering 01/2013; 21. DOI:10.1016/j.ymben.2012.12.003 · 8.26 Impact Factor