We are purifying a protein that is insoluble when expressed as native sequence and in the presence of His-tag. We found a couple of tags, DsbA and ZZ, which are able to make it expressing in the soluble fraction, but as soon as we try to cleave the tag the protein precipitates again. Does anybody know cases of structural and biochemical determination of a protein that retains its fusion partner?
This article is a few years old, but still helpful in my opinion:
Crystal structures of fusion proteins with large-affinity tags
http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/12824478/
It could be that the protein is totally wrong folded. Especially if it exists normally as soluble monomer in the cell and if you express it in a heterologous system.
Thank you for the answers.
Marco, I know that protein mis-folding could be a risk. However, the protein does not seem aggregated when expressed as a fusion protein, from gel filtration. I was wondering if it makes sense to go ahead with preliminar characterization, determining the MW and the secondary structure by CD, DNA binding assays (it is a transcription factor), etc to see if there is structure and activity.
This is a nice review paper that describes the use of protein engineering for protein solubility and crystallization (http://www.ncbi.nlm.nih.gov/pubmed/20445236)
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