CHO Laboratory report – second draft.docx – Engineering of lipid-metabolism in CHO cells by overexpression of SCD1 Abstract Introduction Overall aim is

Engineering of lipid-metabolism in CHO cells by over-expression of SCD1AbstractIntroductionOverall aim is to investigate how SCD1 over-expression influences target recombinant protein expression, as well as how temperature changes may add on to this influence in any capacity. Overexpression of the enzyme Stearoyl CoA desaturase 1 (SCD1) in Chinese Hamster Ovary (CHO) cells, localised in the endoplasmic reticulum, has been demonstrated to enhance secretory recombinant protein yields in several different model biotherapeutic proteins (Budge et al., 2020). The expression of SCD1 is controlled by one of the lipid metabolism modifying (LMM) genes (SCD1). SCD1 catalyses the conversion of saturated fatty acids (SFA) to monounsaturated fatty acids (MUFA), and an increase in the MUFA:SFA ratio results in increased membrane fluidity and cell signalling. Additionally, SCD1 can also directly and indirectly influence the activity of acetyl CoA carboxylase (ACC) which is a rate limiting step involved in de novo lipogenesis. In summary, the activity of SCD1 can increase the rate of lipid biosynthesis and enhance both cell survival and proliferation rate of the cell. Another factor influencing the balance of lipid biosynthesis is temperature. It’s been shown previously that subjecting CHO cells to mild hypothermic conditions (32°C), increases the levels of unsaturated fatty acids (Roobol et al., 2011), which further increases membrane fluidity. As unsaturated fatty acids compress due to decreasing temperatures, the “kinks” in their tails push adjacent phospholipid molecules away, maintaining spaceand increasing membrane fluidity. Need to summarise results section – what other aspects were associated with increased secretory recombinant protein expression. We set out to investigate whether over-expression of SCD1 in CHO cells would increase recombinant protein expression in two different proteins (eGFP and Etanercept). Additionally, we investigated whether cell culture temperature conditions (32 & 37°C) had any additional effect on the SCD1 engineered cells. By transiently transfecting CHO cells with two different plasmids, one containing the SCD1 gene and the other a recombinant protein, we show results.

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