Protein expression in S.cerevisiae - (Feb/28/2008 )
Hi there,
I work with a S. cerevisiae knockout strain which lacks a certain enzyme activity. I want to use this strain to verify the function of the same enzyme from a different eukaryotic species. I transformed yeast with plasmids containing the yeast enzyme (positive control) or the protein of interest. The negative control is vector only. I induce protein expression by adding galactose and harvest the cells when they reach OD=1. At OD=1 the cultures just enter the exponential growth phase. Stationary phase is reached at OD=8.
I performed enzyme assays and found that the positive control showed an activity whereas the protein of interest didn`t catalyze the reaction.
I wonder if the proportion of recombinant protein would rise if I incubated the cultures to mid-exponential phase?
I assume - because of codon usage problems - that my protein would be translated at a slower rate than the yeast enzyme.
Thanks for your ideas!
I work with a S. cerevisiae knockout strain which lacks a certain enzyme activity. I want to use this strain to verify the function of the same enzyme from a different eukaryotic species. I transformed yeast with plasmids containing the yeast enzyme (positive control) or the protein of interest. The negative control is vector only. I induce protein expression by adding galactose and harvest the cells when they reach OD=1. At OD=1 the cultures just enter the exponential growth phase. Stationary phase is reached at OD=8.
I performed enzyme assays and found that the positive control showed an activity whereas the protein of interest didn`t catalyze the reaction.
I wonder if the proportion of recombinant protein would rise if I incubated the cultures to mid-exponential phase?
I assume - because of codon usage problems - that my protein would be translated at a slower rate than the yeast enzyme.
Thanks for your ideas!
I think that you should perform a western to show that you are expressing the protein at comparable levels to your positive control. If your protein levels aren't equivalent, then you may want to grow the cells longer or purify the protein.
Unfortunately, the proteins do not contain a tag and antibodies are not available as well, so that WB is not an option. Do you think I can let them grow until OD of 4 or something like that?
Chalet2
Chalet2
You can always try it and see, but if you get a negative result it's not really telling you anything because you don't know if your protein is being expressed. You could try to do RT-PCR to determine if the transcripts are made at equivalent levels. This won't directly tell you if the protein is translated and folded properly but at least it would give you some idea of what is going on. Alternatively you might want to think about remaking the construct with a tag. Unfortunately, I think that in such an assay, only a positive result is going to tell you anything. A negative result may simply mean that your enzyme doesn't work in yeast (perhaps it lacks cofactors needed for activity, etc.), but doesn't necessarily mean that it doesn't have enzymatic activity.
good luck
smu
Chalet2
western would be the most direct assay to figure out the problem. here is some penny I can throw:
1. whether both the positive control and the target gene are cloned on the same vector? Whether both of them are expressed by Gal promoter? if only the target gene are using gal promoter, then you should double check your strain to confirm it's a gal+ strain. Otherwise it can't be induce the expression in galactose medium.
2. there are many mammalian proteins became unstable in yeast system. That's why ppl here are suggesting you to do the western. If you can't make it, try to express your target gene in a proteinsome defect strain (such as pre1-pre2- or pre4-). If it's a secret protein or membrane protein, you may want to try some lysosome enzyme defect strain such as cpy-. Those stain blocks the potential degradation in yeast.
Again, I suggest you to tag the enzyme and try the western.