If you have a biophysical way to confirm functionality and/or folding it would go a long way towards helping you make this choice.
Does your domain bind a ligand of any kind? Is it significantly stable in a simple thermal melt experiment (of course you have to account for the melting of the fusion partner)? What are the symptoms of precipitation or "disappearance" upon cleavage? How far did you explore the buffer composition for the cleaved material? I have seen a whole spectrum of situations like yours from a nasty misfolded protein kept afloat by fusion, to a perfectly folded domain that simply did not like the buffer conditions and was essentially insoluble after cleavage due to its bull properties' mismatch with those of the solvent. Artem On Mon, Mar 15, 2021, 5:08 AM Schreuder, Herman /DE < herman.schreu...@sanofi.com> wrote: > Dear Bulletin Board, > > Sorry for the slightly off-topic question, but we are struggling with a > receptor domain that expresses well as a fusion protein, but gets lost the > moment it is cleaved from the fusion partner. It could be that the receptor > domain is not or misfolded, but it could also be a solubility problem. > > > > I have seen some crystal structures of fusion proteins with MBP and for > membrane proteins, T4-lysozyme fusions are often crystallized. What is your > experience? Would it be worth trying to express and crystallize a fusion > protein, or would it be better to look for other constructs, e.g. to > include more receptor domains? > > > > Thank you very much for your advice! > > Herman > > > > > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
