I missed the Triton - that will be it! On 7 June 2017 at 15:46, Bonsor, Daniel <dbon...@som.umaryland.edu> wrote:
> It will either be two things. DNA or residual Triton-X-100. When you say, > cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the > pellet and then centrifuged again? If the latter, try sonication. I wash my > IBs at least 4 times with the following buffers; > > > > 1. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5 > > 2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5 > > 3. 10mM Tris, 1M NaCl > > 4. 20mM Tris, 500mM NaCl, pH 7.5 > > > > By resuspension and then sonication. This I find removes DNA and > Triton-X-100. > > > > Also, if the pellet is very large, you may need to increase the number of > washes, volume and length of sonication or split the pellet up. > > > > Other things to try… > > 1. Change the wash salt to KCl and use more, (3M). I was informed > that KCl is a better disrupter of DNA than NaCl (I stand to be corrected if > this is wrong). > > 2. At each wash stage, dissolve a small amount of IBs and measure > the 260/280. The ratio should decrease in the latter washes, if they are > working. > > 3. Does your exonuclease typically contain a divalent metal? You > could try adding EDTA to the wash steps which may help in preventing DNA > stick to your protein. > > > > All the best! > > > > Dan > > > > > > Daniel A Bonsor PhD. > > Sundberg Lab > > Institute of Human Virology > > University of Maryland, Baltimore > > 725 W Lombard Street N370 > > Baltimore > > Maryland > > MD 21201 > > Tel: (410) 706-7457 > > > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Mohammad > Khan > *Sent:* Wednesday, June 07, 2017 9:37 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Problems with an exonuclease > > > > 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. > > > > > -- Best wishes Prof. Jon R Sayers, FRSB Tel: +44 (0) 114 2159552 Email: j.r.say...@shef.ac.uk http://www.sheffield.ac.uk/iicd/profiles/sayers
