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.
