Probably nucleic acids. Increase the number or volume of washes and improve the washing of your inclusion bodies. Instead of sonication, we use a Polytron homogenizer to resuspend the IBs pellet during washing. This is faster and easier. Incorporate an additional chromatography step such as Heparin-Sepharose or Cation-Exchange with SP-Sepharose if the pI of your protein supports this. Alternatively, try to flow your protein through DEAE or Q-Sepharose in the presence of a moderate concentration of NaCl (e.g. 200-250 mM), where much of the nucleic acids should be captured.
- John On Wed, Jun 7, 2017 at 9:36 AM, Mohammad Khan <mohdkhan0...@gmail.com> wrote: > 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. > >
