Inhibition of the ubiquitin ligase activity improves the production of biologically active fusion protein HSA-HGF in Chinese hamster ovary cells.

ABSTRACT: Hepatocyte growth factor (HGF) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. The aim of this study was to enhance the expression of recombinant fusion protein HSA-HGF (R494E) in CHO cells by inhibiting the intracellular ubiquitin ligase activity. The high stable expression sub-clones with different signal peptides were selected by western blot (WB) analysis and used for suspension culture. We found that the expression of fusion protein HSA-HGF (R494E) on day 3 achieved 50 mg/L during the 8 day culture process, a large number of fusion proteins were intracellular degradated by ubiquitination pathway during day 4 to day 8. Furthermore, ubiquitin ligase inhibitor, thalidomide, was added in culture process, and resulted in efficient and stable secretion of HSA-HGF (R494E) in CHO cells. According to biological activity assays, HSA-HGF (R494E) possessed various biological activities similar to native HGF. In conclusion, innhibition of intracellular ubiquitin ligase activity was successfully improve the expression of biologically active fusion protein HSA-HGF (R494E) in CHO cells. Our data may be beneficial to enhance the production of other therapeutic proteins in fed-batch culture.

SUBMITTER: Xu D 

PROVIDER: S-EPMC5470524 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature