Hi,
I was carrying out Galactose induction experiments to determine protein stability. However, one of my wild-type strains expressed only a trace amount of proteins, which was unexpected as galactose over-expresses proteins.
I confirmed the presence of the plasmid that carried my gene of interest using a slight variation of the experiment. Protein levels were observed, so there was nothing wrong with the plasmid. But I'm not sure why the proteins were not expressed in Galactose?
Can someone explain to me why this would happen?
Is the gene of your interest from yeast? If not, you may want to check the codon usage, and get it codon-optimized for expression in yeast. Even so, you may not always see high levels of induction for all the proteins you would like to test. For some reasons, certain proteins may not be expressed at high levels, even if you put them under a strong promoter.
Hi Demagi,
did you achieve good levels of biomass in your wild-tipe culture?
You could tried to optimize the culture for example enhancing the aireation rate, which is critical for yeast, or changing the way of gal-induction by feed-batch.
Nagendra Nath Das
I doubt so, as other gal induction experiments worked, and protein levels were observed.
Hua Xiao thanks for the reply. The gene of interest is a plasmid-borne allele introduced to a yeast strain background. It was odd that my Wild-type strain didnt show protein levels and my mutant strains did. Not sure why that was? But yes, maybe it wasnt expressed even with a strong promoter, because my protein acts as a repressor itself.
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