Recombinant protein production remains a central component of many biotechnological projects. As mammalian proteins frequently require extensive post-translational modification, mammalian cells are often used for recombinant protein production instead of yeast and bacteria. Intuitively, a protein will be best expressed in its native cell type under physiological conditions, where a multitude of
molecular systems work together for efficient production and quality control at various stages, including synthesis and folding, post-translational modifications and subcellular targeting. However, as mammalian cell culture is costly and establisment of efficiently producing cell lines can be a lengthy procedure, productivity is an important determinant.
Researchers from the LBI-CR have now reported highly efficient vectors for protein production with an optimal chromatin environment based on Bacterial Artificial Chromosomes (BAC) with open chromatin regions. These vectors have been established as valuable tools for recombinant protein production in Chinese Hamster Ovary (CHO) cells, the preferred mammalian cell type for idustrial protein production. Since they confer position independent, copy number dependent and high and stable expression and can also be generated very quickly, these BAC-based expression vectors provide a fast and efficient alternative to conventional cell line based production methods.
Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. Out of several constructs we identified the Rosa26
BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.
We therefore show that BAC based expression vectors can support protein production in CHO cells impproved by an order of magnitude compared to conventional vector systems. This vector system has tehrefore the potential to impact positively on cost effienciency of any commercial recombinant protein production.
This report was now published in Nucleic Acids Research