Dear all, I am working with an exonuclease by refolding it from inclusion bodies (IBs). I tried various constructs and hosts, but couldn't get it in soluble form.
I lyse my cells using a cell disruptor and after solubilizing IBs with urea, I refold the protein by rapid dilution and get an aggregate and monomer peak of the same on GFC. and have checked CD as well as activity, both of which are good. My issues is as follows: I get a high 260 nm peak while purifying it on GFC. the 260/280 ratio can reach upto 2. I have tried all means to get rid of watever this contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase prior to lysis. I have also used methods to remove the DNA from protein, if that is the contaminating agent. I am trying to crystallize the protein with no success so far. Moreover, my thermofluor assays give very low fluorescence. I use Sypro Orange as a fluorophore. Suprisingly, a point mutation in the active site (His to Arg) gets rid of the issue of contamination and gives me good thermofluor curves. I purify the mutant also form IBs. Can someone suggest what this "contamination" may be? Thank you for your time.
