I intend to monitor post-translationally modifications on a protein with very low expression in yeast. I have already upscaled cell culture volume and done immunoprecipitation with considerable antibody concentration for protein enrichment but the amount of protein recovered is not suitable yet to do MS. Any suggestions to move a step forward on my procedure?
Did you try grow your yeast on differents carboun and nitrogen source? Because, sometimes, microrganisms may be under catabolite repression.
Please look at the expression construct and see if they have rare codons for yeast expression. Correct it if possible.
reclone your protein while including two things upstream of your start codon:
1- Yeast ribosomal binding site.
2-Promoter of a house-keeping gene.
As Wael pointed out, introducing a consensus Kozak sequence 5' of the ATG start codon (at least AAA-ATG) may help improve translation.
If you are trying to maintain this protein under the control of its endogenous promoter, you should be able to subclone the gene with its regulatory sequences into a 2 micron plasmid that should increase the protein level considerably.
Another idea would be to try expressing a fusion of your protein to a tag designed specifically for affinity purification such as Protein A or Strep tag II. You should be able to integrate the tag sequence into the genome using PCR and a suitable pFA6a-based plasmid.
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