I am expressing a mammalian protein in the yeast H. polymorpha however, despite transforming the chaperones required for proper protein folding, the expression of the protein is not attained. What strategies/changes can be employed?
how have you evaluated the expression? Western blot?
it is possible that your western blot had an issue. Sometimes it’s easy to mess up your western blot samples and have the proteins degrade.
I suggest:
1. As a control, transfect mammalian cells with the plasmid for this protein. Get the cell lysates from that. Retransform your yeast (just redo it all). Get the cell lysates from that. Now you have cell lysates from
1. Untransfected mammalian cells
2. Transfected mammalian cells
3. Untransformed yeast
4. Transformed yeast.
run the western blot with these, also make sure you stain for housekeeping genes to assess the quality of your protein samples and make sure the western is working. See if you are getting a band for just the transfected mammalian cells. Then you know it’s a yeast producing the protein issue, maybe a chaperone expression issue. If you get a band for both, great. If you get a band for neither:
you need cell lysates from cells that express the protein natively. This is the best positive control.
run western with that and see if you get a band: if you do, then you know that it’s an issue with the plasmid. If you don’t, then it’s an antibody issue.
Youve got this. Best of luck. It’s going to all work out!
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