HI Savvas, We recently had a protein that showed two overlapping peaks on the disposable fast flow Q columns, so we decided to see if we could resolve them with a higher resolution Q media. It ended up having 7 distinct peaks, only one of which was free of contaminants. We have also noticed that the presence of heat shock protein bound to our favorite protein is highly dependent on the induction time/temp, and also varies between bacterial strains. It is also effected by the media. Yo might try osmotic shock or other additives in the media.
We have also had luck by adding 1-3 molar urea in the lysis buffer. We get lots more protein out, with better purity, but some of it crashes, which I think is purifying out the misfolded protein. Lastly, you might try a fusion protein to something that has chaperone activity, like MBP, which may mask the binding epitopes for the other proteins. Best regards, Kendall Nettles On Apr 19, 2011, at 3:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. ---- Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
