Several further notes after contemplation, lunch and a slow day. If the protein is His-tagged, you could stick the unfolded protein to Ni-Resin and extensively wash with 8M Urea to remove DNA/Triton-X-100, elute, and then refold. This may be easier than multiple sonications/centrifugations. Or you could stick the folded protein that you have purified to the Nickel resin and wash with high concentrations of salt to remove DNA/Triton-X-100.

To check if is really DNA-protein complex you have prepped, you can run two agarose gels. Stain one with ethidium bromide and the other one coomassie stain. The two bands should coincide with each other (protocol for native agarose gel electrophoresis can be found here http://www.sciencedirect.com/science/article/pii/S0003269700945986).

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:[log in to unmask]] On Behalf Of Mohammad Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: [log in to unmask]
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.