Transient gene expression (TGE) is based on episomal DNA expression. This technique is used for the production of recombinant proteins that can be used as therapeutics. TGE is for example extremely useful for shortening the time between candidate proteins screening and their production. Understanding and optimizing TGE is a major concern in the field of recombinant proteins. Thus, we selected here two different approaches to further improve TGE. First, we studied effect of signal peptide (SP) modification on TNFR-Fc protein in transiently transfected HEK293E and CHO-DG44 cells. Secondly, we studied effect of Valproic acid on the production of TNFR-Fc protein in HEK293E cells and on its influence on plasmid methylation. Modification of TNFR-Fc was done with eighteen others SPs. Artificial SPs exhibiting different scores using a prediction method and SPs coming from naturally secreted proteins were tested. None of them could induce a better secretion than the native SP. Nevertheless, they all showed comparable secretion abilities, except one that could not induce protein secretion. We showed here that SP modification could not increase protein secretion in HEK293E or in CHO-DG44. But also that without a valid SP, protein secretion is inhibited. Addition of VPA in HEK293E cells transfected with pXLG-A3 plasmid increased IgG productivity by 11 fold. Nevertheless, we showed here that VPA did not increase TNFR-Fc productivity to the same extent (1.2 fold) even by optimization of its concentration. Furthermore, we investigated the effect of plasmid methylation (TNFRFc and IgG) on TGE using VPA. We found that IgG expression was less influenced by this modification than TNFR-Fc. In fact, TNFR-Fc plasmid methylation drastically decreased specific productivity (0.2 fold). And VPA could almost entirely compensate this effect (0.8 fold). We therefore hypothesized that VPA has an impact on pDNA methylation in the context of TGE.