If using 2% hot SDS to extract laccase from yeast cell wall, dose it denature the laccase? This is my first time extracting protein. Could you give me some suggestions about this?
How hot is the sds solution? I think it will denature your protein either by temperature or by sds action.
That's too harsh. Do you have an idea on how the proteins are interacting with the cell wall? If it is only ionic you can compete for the interaction to release the protein. Another thing you can explore is enzymatic hydrolysis of the cell wall.
The conditions are indeed harsh, but this doesn't mean that active protein cannot be recovered. I have refolded proteins that were isolated from purified inclusion bodies by SDS extraction followed by extensive dialysis to remove the SDS and then purification by IMAC with on-column refolding.
Alternatively, you could try using a protectant additive. This has been successfully used for SDS-extraction of GST-tagged proteins that retained activity following purification - see the Biotechniques paper by Elodie Boisselier et al :
https://www.ncbi.nlm.nih.gov/pubmed/21906042
PMID: 21906042 DOI: 10.2144/000113739.
I used an adaptation of the above to isolate proteins extracted in the pseudo-native fraction using n-lauroyl-sarcosine that retained GST-resin-binding capability, so it is clearly a method that can be used flexibly with some care if you have time to trial the conditions.
Good luck!
Would Triton X-1000 and CHAPS be enough to lyse the cell wall? I haven't worked with fungi but that's what I used in animal cells for non-denaturating solubization of proteins.
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