Optimising recombinant protein expression level in Saccharomyces cerevisiae - (Jan/03/2012 )
Hi, I am working on the expression of recombinant protein in S. cerevisiae. The system I am using is pYC2/CT from Invitrogen, which contains a GAL promoter for inducible expression.
Does anybody know by varying the galactose concentration in the induction medium could contribute to the variation in expression level? (as we varying IPTG concentration in E. coli expression). The user manual only says to use a 2% galactose concentration for induction.
Anybody can help me? I have expressed my protein using the GAL promoter system above. No enzyme activity and not detected in Western blot. I wonder what are the problems. Below are some details of my experiment:
1) the expression construct had been sequenced and contain no mutation. (and therefore no frameshift)
2) the positive transformants (in my case S.cerevisiae) were selected from minus uracil agar and confirmed by PCR colony.
3) Induction using 2% galactose for 24 hour. (and raffinose as C source instead of glucose)
3) Protein extracted using the commercial kit known as Yeastbuster reagent.
4) On SDS-PAGE i can see some smearing at the target size in which the control (host) do not have. And the smearing could due to the diff level of glycosylation in s.cerecisiae.
5) the positive control for Western (which is B-galactosidase) was not detected. But a control using a protein fr my friend turned out to be a nice band.
Any comments or ideas? I do not know what to do...