Hi,
I am trying to secrete a heterologous protein in S. cerevisiae using alpha mating factor signal peptide at the N-terminus and GFP at c-terminus. The plasmid is a 2 micron (Ura selection) plasmid having Gal promoter. After induction at 30 degree (2% galactose), I have collected time points up to 24 hrs and separated the media and cells. The media was TCA precipitated and washed with 80% acetone for SDS-PAGE analysis. I see no protein on the gel. Is there any problem with my processing? Is the induction period sufficient? Has anyone tried this approach? If yes, what was the yield of the protein?
note: my protein is 83 kDa... so in total--- alpha mating factor+protein of interest+GFP = approx 120 kDa protein
Kex2 and ste13 sites were included during cloning.
thanks in advance.
Hi there,
Is your protein actually expressed and not properly processed? You should look at the cell content before and after induction. On what sugar do you grow the cell before induction? As glucose is inhibitory to gal driven expression, cells have to be washed from glucose prior to induction. Lag phase before induction is dépendent on the amount of glucose still present in cells. The best is actually to grow cells on raffinose so galactose induction will drive proper expression as raffinose is not inhibitory to gal driven expression.
Dear Dr. Dominique Liger,
I grow my cells in glucose and wash with sterile water before putting on galactose. I have expressed the very same protein intracellularly and confirmed by microscopy and western. I want to secrete it out so that purification will be easy.
Hi again,
Do you know if the protein is expressed but just not secreted? The issue is actually not the same If there is no expression at all or if there is expression but no secretion.
Hi,
I checked under microscope, but the signal was weak. So am not sure if my protein is expressed, but I have complementation assay which scores for expression of functional protein. the intracellular expression construct is positive in complementation experiment whereas the secretory system is expected to be negative. But I see that the secretory system also is mildly positive (growth seen in dilutions containing higher OD of cells) but not to the same extent as intracellular one. In short, its growth is better than negative control (empty vector) but way lower than intracellular expression. Since the growth is conditional to the expression of my protein of interest i inferred that protein is getting produced but some of it is stuck inside the cell.
Well the problem is that complementation doesn't require a lot of functional protein and does occur in cytoplasm, whereas you are looking for fully processed/secreted protein for purification (ie. a lot more than for complementation)...
Hi -
As per Dominique, you'll need to switch your carbon source prior to induction - . Recommend these protocols:
When you wash your Yeast Cells before induction - I'd use minimal medium without any sugar supplementation.
Protocols for Gal Induction in Yeast
http://www.bio.brandeis.edu/haberlab/jehsite/pdfs/GALInduc.pdf
http://capricorn.bc.edu/bi204/wp-content/uploads/2014/07/13-protein-overexpression-2014.pdf
Glucose repression of Gal Induction in Yeast
http://web.mit.edu/7.03/documents/7.032006Lecture22.pdf
Good Luck - Looking Forward to Hearing about your Success!!
Dear Dr.Dominique Liger and Dan Weaver,
Thank you for your responses. I will go through the references you have suggested.
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