| High protein titer is a desirable trait of mammalian cell cultures. Different approaches have been applied over the years to increase product concentrations in bioreactors. Analysis of the molecular characteristics involved in two of the approaches, metabolic shift and butyrate induction, were the focus of this thesis. Cultures that undergo metabolic shift attain higher cell concentrations, and thus, higher protein product concentration. Butyrate enhances the cellular specific protein productivity.; Metabolic shift as well as histone acetylation effects are the manifestation of changes in environmental factors, activities of enzymes/transporters, and translation and transcription of numerous genes involved. One cannot possibly exhaustively investigate every detail in the multi-layered regulatory network that may be involved in these processes. Genomic and proteomics techniques have been used to better understand the molecular mechanisms involved in metabolic shift and butyrate effects on enhanced protein production. These tools, in contrast to conventional methods, allow for a global survey.; The effects of butyrate on gene expression were more drastic than metabolic shift. The molecular response to metabolic shift was concentrated in the glycolytic pathway. There is a strong possibility that the metabolic shift is dictated by the interaction of the cells with their environment. Butyrate exposure led to a more widespread gene alteration effect, including pathways such as protein folding, cell growth, apoptosis, and stress response. Some genes changed similarly in both systems, such as cyclin D2, KDEL endoplasmic reticulum protein retention receptor 3, and lactate dehydrogenase. Genes known to be activated during plasma-cell differentiation, such as genes involved in the unfolded protein response, were induced by butyrate exposure. Plasma cells are natural super secretors.; The processes that lead to substantial protein productivity increase were accompanied by a relatively small extent of differential gene expression, although, the number and complexity of the interactions between the genes involved was high. It is very unlikely that perturbation of only one gene will make the goal of engineering a high-producer cell achievable. It became clear that a group of genes that act in concert, such as during plasma-cell differentiation, would make this goal more realistic. |
