No abstract

It is widely reported that the growth of recombinant bacteria and yeast is adversely affected by increased metabolic load caused by the maintenance of plasmid copy number and recombinant protein expression. Reports suggest that recombinant mammalian systems are similarly affected by increased metabolic load. However, in comparison to bacterial systems relatively little information exists. It was the aim of this study to test the effects of recombinant gene expression on the growth and metabolism of two industrially important cell lines. A BHK and CHO cell line were stably transfected with the human gastric inhibitory peptide (h-GIP)and glucagon receptor respectively. Selection was by way of the neomycin resistance (neo (r)) gene using G418.The growth and metabolism of both cell lines was affected by the presence of G418 in a manner indicative of increased metabolic load and which appeared to be caused by over-expression of the neomycin resistance protein. The two cell lines differed in their metabolic response to G418, which suggested that some cell lines or clones may be better able to tolerate a metabolic load than others. Growth under increased metabolic load was affected by medium composition with serum, insulin and glutamine concentration as influencing factors. Implications for the use of G418 are discussed.

In a previous article (Yallop and Svendsen 2001), recombinant CHO and BHK cell lines, expressing the human glucagon receptor and the gastric inhibitory peptide receptor, respectively, showed reduced growth rates and altered nutrient utilisation when grown with increasing concentrations of G418. This response was associated with an increased expression of the neo (r) protein, while expression of the recombinant membrane receptors remained unaltered. The metabolic response was characterised in both cell lines by an increase in the specific rate of glutamine utilisation and in CHO cells by a decrease in the yield of lactate from glucose, suggesting a change in the flux of glucose through central metabolism. The aim of this study was to further elucidate these metabolic changes by determining the activity and relative expression of key enzymes involved in glucose and glutamine metabolism. For both CHO and BHK cells, there was an increase in the activity of glutaminase, glutamate dehydrogenase and glutamine synthetase, suggesting an increased flux through the glutaminolysis pathway. The activity of glucose-6-phosphate dehydrogenase and pyruvate carboxylase in CHO cells was also increased whilst lactate dehydrogenase activity remained unaltered, suggesting an increased flux to the pentose phosphate pathway and TCA cycle, respectively. The activity of these enzymes in BHK cells was unchanged. Quantitative RT-PCR showed that expression levels of glutaminase and pyruvate carboxylase were the same with and without G418, indicating that the differences in activities were likely due to post-translational modifications.

No abstract

The maintenanee of high plasmid eopy-number and the over-expression of foreign proteins in reeombinant bacteria and yeasts is known to resuh in a metabolie load, whieh adversely affects the growth rate. Reports suggest that recombinant mammalian systems are similarly affected, however in eomparison to bacterial systems, relatively little information exists. It was the aim of this study to test the effects of reeombinant gene expression on the growth and metabolism oftwo industrially important mammalian eell lines. BHK 570 and CHo..Kl eells were stably transfected with the human gastric inhibitory peptide (h-GIP) and glucagon reeeptor respectively. Selection was by way ofthe neomycin resistance (neo') gene using G418 as the selective agent. On removal of G418, production of both receptors was stable for the course of the experiments. The growth and metabolism of both cell lines was affected by the presenee of G418 in a manner indicative of an increased metabolie load, eaused by over-expression of the neo' protein. The two eell lines differed in their response to the metabolie load, suggesting a cell-line or clone dependent response. Growth under the increased metabolie load could be modulated by serum, insulin and glutamine addition to the growth medium. Implications for the use of G418 are discussed.