Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters tested. Secondly, 5 differ... More
Efficient production and secretion of recombinant proteins in mammalian cell lines relies in a combination of genetic, metabolic and culture strategy factors. The present work assesses the influence of two key genetic components of expression vectors (promoter and signal peptide) on protein production and secretion effciency in HEK293 cells expressing eGFP as a reporter protein. Firstly, the strength of 3 different promoters was evaluated using transient expression methods. Flow cytometry analysis revealed that the highest level of intracellular protein expression was found when eGFP was under the control of CMV promoter, being 3-times higher in comparison to the rest of the promoters tested. Secondly, 5 different signal peptides were assessed in stable transfected cell lines. Spectrofluorometry was used to determine intra- and extracellular protein expression levels in terms of fluorescence, and the results were further confirmed by SDS-PAGE. The highest secretion efficiency was found for human IFNα2 signal peptide, achieving up to 2-fold increase in the amount of secreted protein compared to other signal peptides. The results showed that the combination of CMV promoter and IFNα2 signal peptide resulted highly efficient for recombinant protein production in HEK293 cells.
