Genetic manipulation of the protein synthetic capacity of mammalian cells

Major improvements in the yield of recombinant proteins produced by animal cell cultures are likely to result from an extension of productive cell lifetime. Genetic engineering of cell cycle and death mechanisms and /or improved fed-batch processes have been used to enhance cell productivity. In contrast, by direct genetic manipulation of the rate of protein synthesis in animal cells we intend to augment both cell specific production, and productive cell lifetime in batch culture. This approach also provides a means to increase the transient expression of recombinant proteins by ‘transient’ host cell engineering. mRNA translation initiation factors control the overall rate of protein synthesis. The phosphorylation state of a subset of these factors; 4E-BP1, eIF2B, eIF4G, eIF2 and eIF4E are important in the regulation of protein synthesis (Figure 1.). An increase in the phosphorylation state of the a subunit of eIF2 is associated with an inhibition of protein synthesis in response to stimuli such as heat shock (Duncan & Hershey, 1987), amino acid, glucose, or serum deprivation (Scorsone et al., 1987). The impairment of translation seen in response to heat shock is partially overcome by expression of a non-phosphorylatable mutant of eIF2?, Ser5lAla (Murtha-Riel et al., 1993). Thus, reduced rates of protein synthesis seen when cells encounter other such conditions are also expected to be attenuated by expression of this mutant, which is otherwise functional in translation

Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering