I already know the target protein is rapidlly degraded in yeast cells, and this degradation is dependent on the ubiquitin-proteasome degradation pathway. I want to konw how to increase my target protein's stability.
Dear Ronghua,
It is not entirely clear how you would like to achieve stabilisation of your protein. You say that you know your protein is degraded through the ubiquitin-proteasome pathway. If you know which lysine(s) is(are) targeted for ubiquitylation, you can mutate these to arginines. This will abolish their ubiquitin modification and also subsequent proteasomal degradation (assuming this is entirely ubiquitin dependent). Another option is to use a strain deleted for the E3 ubiquitin ligase of your protein of interest, which should lead to stabilisation, if there is no redundancy. Lastly, you can use inhibitors of the proteasome to block degradation - usually it is necessary to use specific yeast strains with deletions that favour permeability and leads to less drug efflux (erg6Δ, pdr5Δ) to achieve a good inhibition by e.g. the MG132 inhibitor.
Good luck!
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