Dear all, I apologize that till now I was making the mistake of not sending the replies in the common thread, but somehow only to each person individually. I am summarizing my answers since yesterday here:
I have tried refolding it at pH 7, 7.5 and 8. I add 4mM TCEP to my urea buffers and reduce it to 2 mM on refolding. I have tried in presence of DTT and also in absence of any reductant. My refolded wild type enzyme shows activity, whereas the mutant doesn't. Yes, I have got my protein mass-speced. Didn't see any modification! I have tried to remove the contamination by affinity and ion exchange but with no avail. I have to try heparin-sepharose though. I sonicate my samples in the washing steps. I also suspected Triton X as the contaminant. But I then figured out that TX-100 has a high absorbance at 280 nm, rather than at 260. I will surely try the KCl and EDTA suggestions. I have also tried purifying in absence of TX-100, but with same results! As for running an agarose gel, I have done so but couldn't really see anything on an EtBr gel. I will try the suggestions of washing with other solutions as suggested. Thank you all for the great suggestions! On Wed, Jun 7, 2017 at 3:36 PM, 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. > > >
