| Recombinant proteins reprent a group of biopharmaceutical products showing the fastest growing pacein the current biomedicinal industry. Their production depends on variouscell substrates, like E.coli,yeastand mammalian cells. Among the mammalian cells, the CHO cellhas emerged as the most popularone in terms of both human use and commercialization. CHO cells can not only ensure proper folding, packaging and modification post translation, but also produce much higher quantity of foreign proteins with the biological activities much closer to native proteins. In addition, the CHO cells can be easily adapted from adhesion growth to suspension growth.However, even though these features, there are still many bottleneck factorslimiting the capacity of CHO cells of further improving quantity and quality of recombinant proteins. One of the major bottleneck factors comes from the sensitivity of the cells to apoptosis occurring commonly during the prolonged cell culturing or special treatments. Therefore, theoretically, it is possible to improve the productivity of recombinant proteins in CHO cells by enhancing the anti-apoptosis activitity of the cells. Based on this hypothesis, this study was designed to engineer CHO-K1 cells to generate the apoptosis-resistant cells in order to achieve the increased productivity of recombinant proteins.In this study, we employed overexpression of human Bcl-xl(h Bcl-xl) gene as the major engineering technology to increase the anti-apoptosis activity of the cells. h Bcl-xl is a key anti-apoptotic member of the Bcl-2 family proteins and can modulate apoptosis via different molecular mechanisms. We first established the CHO-h Bcl-xl cell line, which was integrated with and stably expressed h Bcl-xl, and then tested the anti-apoptosis activity of the newly generated cell line to both physical(UV light) and chemical(mitomycin C) apoptosis inducers. Next, we transfected the EGFP gene into the CHO-h Bcl-xl to establish the h Bcl-xl/EGFP cell line, which was used as a model cell line and the expression of EGFP was used to represent the expression of recombinant protein, for the evaluation of the effect of h Bcl-xl on both expression and structural stabilities of recombinant protein during apoptosis induction or the treatment with sodium butyrate(Na Bu). The evaluations were performed along the entire process including cell expansion on adhesion growth, adaptation to suspension growth, and establishment of both Master Cell Bank(MCB) and Working Cell Bank(WCB). Our results showed that the stable over-expression of h Bcl-xl at all stages of the entire process significantly increased the resistance of CHO cellsto apoptosis induced by UV light, mitomycin C and Na Bu. In addition, comparing with control cell line, i.e. the Vector/EGFP cell line derived from CHO-Vector control cell line, the Bcl-xl overexpression maintained a much higher level of both quantity and structural integrity of the EGFP production.More importantly, the stable overexpression of h Bcl-xl supported a dramatic increase in quantity of EGFP expression induced by Na Bu, especially in suspension growth, while still maintaining the structural integrity of the EGFP. Comparing with the cells undergoing no h Bcl-xl transfection and no Na Bu treatment, the compound effect of stable overexpression of h Bcl-xl resulted in over 27-fold increase in EGFP productivity.Based on the present study, it can be conclud that h Bcl-xl overexpression can increase anti-apoptosis activity of CHO cells to different apoptosis inducers and stable expression of recombinant proteins, and most importantly can support the Na Bu-enhanced expression of recombinant protein while maintaining its structural stability. |
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