I am trying to optimize the expression conditions for S. cerevisiae yeast cells, I need high expression for downstream screening, and was wondering if anyone of you has tried higher Galactose concentrations and/ or if any other modification has helped to increase the concentration of the expressed protein? I have co-expressed two genes in pESC-His, with Gal1 and Gal10 promoters, the protein from gene one acts on the protein from gene two and makes a cyclic peptide. I have tried many different time points of induction, different sugar source (glucose, galactose, raffinose) to grow cells to OD600 = 1.0 and then induce them with 1%, 2%, 3% synthetic galactose medium, however, I see very little increase in the expression say from 4ugs/100ml to 6.8ugs/100ml. I need at least 20ugs/100ml...any suggestions would be appreciated! Thanks in advance!
Hi there,
I don't think you can further optimize galactose concentration as 2% is already giving the maximum response. If you need more protein then scale up the experiment (or make more biomass as suggested above).
Thank you Jorg and Dominique, I will definitely try to increase the biomass as suggested by Jorg to see if I see any drastic increase in the expression. However, my aim was to get 20ugs per 100ml , so that we could get 200ugs from 1L culture after scaling up!
Hello Tina,
I am trying to express two different proteins u.nder Gal1 and Gal10 promoters,but they fail to express. After transition of media from YPD to YPG, I have not added any Galactose additionally. Can you please advise?
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