Cell-free coupled <i>in vitro</i> transcription-translation offers many advantages for the production of recombinant therapeutic proteins using the CHO lysates. However, many glycoprotein's are still difficult-to-express using this system. To address these hurdles a variety of different approaches can be taken to obtain the expression outcome desired. Here, we report the effect of air, nitrogen, and oxygen on the overall expression of erythropoietin (EPO), an important endogenous hormone which stimulates the development of red blood cells. Expression of EPO was performed in a dialysis cassette sparged with the gases into the expression system effectively allowed delivery of reactive gases into the cell-free reaction. The results showed that the presence of gases (air or nitrogen) on the buffer side of the expression system enables a redox reaction, effectively increasing the expression of EPO in comparison to other gases tested. We were able to analyze the expressed protein by conducting a western blot analysis, effectively identifying EPO at ~30-37 kDa. The proposed method could provide meaningful insight into developing optimal cell-free reaction conditions for various recombinant therapeutic proteins of interest.

Effect of gases on the expression of recombinant erythropoietin using CHO cell-free coupled <i>in vitro</i> transcription and translation system

2019 Spring National Meeting

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Effect of gases on the expression of recombinant erythropoietin using CHO cell-free coupled in vitro transcription and translation system