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Recombinant Protein Production

Recombinant protein production in eukaryotic cells is one of the main topics of biotechnology. One of the most critical steps is the development of appropriate vectors to express the protein of interest. Bacterial Artificial Chromosomes (BACs) containing the appropriate locus can be used as expression vectors in the protein production field. In collaboration with Dr. A. Bauer, we have developed a BAC-based vector system that improves the protein production by a factor of 10 when compared to a conventional vector in stable HEK293 cell lines. We are currently expanding our vector tool-box and exploring the use in different cell lines, such as CHO cells.

Recombinant protein production using Bacterial Artificial Chromosomes as expression vectors in eukaryotic cells

Recombinant protein production in eukaryotic cells is one of the main topics of biotechnology. One of the most critical steps is the development of appropriate vectors to express the protein of interest. Bacterial Artificial Chromosomes (BACs) containing the appropriate locus can be used as expression vectors in the protein production field. In collaboration with Dr. A. Bauer, we have developed a BAC-based vector system that improves the protein production by a factor of 10 when compared to a conventional vector in stable HEK293 cell lines.

Bacterial Artificial Chromosomes (BACs) are large vectors that can contain up to 400 Kb. They may accommodate an entire locus with all the regulatory elements that control the transcription of a gene. Thus, BACs harboring the appropriate locus contain their own chromatin environment and they are not affected by the surrounding chromatin to their genomic integration site. Due to these characteristics, BACs are largely used in the mouse transgenic field. Interestingly, these attributes make the BACs very attractive expression vectors for recombinant protein production in mammalian cells.

We take part of in the European AIDS Vaccine Initiative 2020 (EAVI2020), where we will use the BAC-based expression system to produce new HIV vaccines. We are trying to improve the BAC-expression system by performing an unbiased screening in CHO cells and identifying hot spots which permit high antibody production based on the genomic environment. Such genomic regions containing appropriate hot spots will be retrieved and tested as BAC-based expression vectors for antibody production in CHO cells. With this approach, we expect to develop an improved series of BAC-based expression vectors that will confer reliably high and stable levels of recombinant protein expression thereby reducing the costs and time to establish appropriate expression systems.

Schematic representation of the vectors used in this work: A) CAGGS Fc: conventional vector containing the CAGGS promoter and the human IgG1-Fc as protein of interest. Rosa26CAGGS Fc: CAGGS Fc vector recombined into a BAC containing the Rosa26 locus (BAC-based vector). B) Protein production in HEK293 stable bulk cultures from a conventional vector (CAGGS Fc) and a BAC based vector (Rosa26CAGGS Fc). The use of a BAC-based vector improves the protein production by a factor of 10.

 

 

 

Bacterial artificial chromosomes improve recombinant protein production in mammalian cells. (A) Clonal distribution of IgG-Fc cell pools shows the specific productivity picogram/cell/day (pcd) of the 20 best clones out of 50 pre-screened clones derived from stable CHO cell pools generated with the Caggs:BACRosa26, Caggs:BACHprt and Caggs:plasmid control vectors. (B) Production of a single polypeptide using a BAC-based vector under Polymun industrial settings. CHO K1 cells were transformed with a BAC-based vector expressing the GP140 polypeptide. The productivity of a GP140-BAC single clone was measured in a fed-batch culture during 14 days. For comparison, a Polymun conventional plasmid-based GP140 producer (DHFR amplified) is shown. The GP140 clone producer generated with the BAC-vector system (GP140-BAC) reached a maximal titer of 1010 mg/l, while the conventional/plasmid-based GP140 producer (GP140 Conventional plasmid) reached a maximum of 6.4 mg/l.

 

Bacterial artificial chromosomes improve recombinant protein production in mammalian cells. Strategies for production of the anti-HIV-1 antibody PG9 as a complex protein model using BAC-based vectors. (A) Schematic view of human IgG1PG9 antibody BACs constructs. In the case of BAC 10, BAC 20 and BAC 30 constructs, heavy chain (HC) and light chain (LC) expression units were inserted into the same BAC Rosa26 at different locations 10, 20 and 30 kb apart from each other (example shows BAC 20). Lower panel, BAC LC and BAC HC vectors: LC and HC expression cassettes were recombined into two independent BACRosa26 vectors. Stable cell clones were generated by co-transfection of these two independent BACs (BAC HC/LC). BAC 2A and BAC IRES: LC and HC expression cassette was inserted into a single BAC. LC and HC expression was linked by a bicistronic mRNA using the FMDV 2A peptide or an IRES element. (D) Specific productivity in pcd of the 20 best clones from 50 pre-screened clones derived from the BAC HC/LC, BAC 10, BAC 20 and plasmid HC/LC (p HC/LC) stable cell pools. n = 2–4 independent transfections, error bars represent SEM. 

 

Funding:

This research is conducted in close cooperation with and thanks to financial support of POLYMUN Scientific Immunbiologische Forschung GmbH and is part of the EAVI2020 project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No .681137.
This cooperation was initiated and was formerly financially supported in part by the Austrian Research Promotion Agency (FFG) through bridge project "BAC-based Expression System Technology" (829611).

 

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