Thanks for the suggestions so far, I should have given a little more information in my initial post. The protein I'm working on is an Gram-Neg Beta-Barrel about 100kDa, likely with 22 strands.
I'm currently crystallising in bOG, although my next optimisation step is to try a range of detergents. I'm using a refolding protocol which relies heavily in LDAO (one of the only cost effective detergents for the volumes I'm using), I refold, nickel purify (N-terminal tag, signal peptide removed) , do a size exclusion step (S200), then detergent exchange into bOG using a nickel column. The BOG concentration is obviously high 0.8-1%, which might be causing the gels? The gels don't look like classic phase separation to me (from soluble proteins that is, this is my first membrane protein), they are not spherical, the tend to float or sit on the bottom and are semi-solid, around 30-100uM diameter. These screens I've set up are classic screens for membrane proteins (mem-plus, mem-start, PGA etc.) and the drops aren't dried up. The gels are strongly birefringent, but the drop is not. Additionally the conditions in my initial screen which yielded crystals seemed biased for the formation of the gels. Thanks again for the help, I feel I might have a long raod to optimisation ahead of me. Rhys ________________________________________ From: Jim Fairman [fairman....@gmail.com] Sent: 09 May 2013 20:58 To: RHYS GRINTER Cc: ccp4bb Subject: Re: [ccp4bb] Membrane Protein Optimisation With information you've provided I have multiple suggestions for you, some of which you may have already tried: 1. OMPs can be fairly particular about which detergents they will crystallize in. Try exchanging the protein into a different detergent/detergent mixture and then set up new trays in your favorite broad matrix screens. DDM, DM, LDAO, OG, and C8E4 are a few of my favorites for OMPs, but there are many others. This can be done using a size exclusion column as the final purification step for your protein where the column is equilibrated with the appropriate detergent. This step will also let you know how well behaved the protein is in that detergent via the shape/height of the peak. This won't help you optimize your current condition, but it may lead to different/new conditions with even better crystals. As Pascal suggested, try to carefully choose which MW cut-off concentrator you end up using. Minimizing the amount of detergent concentration that occurs during your concentration step(s) is optimal. 2. Attempt crystallization using DHCP/CHAPSO bicelles. You can buy them pre-made from MemX Biosciences or make them yourself using a published protocol from David Bowie's lab. These have been used to crystallize the mitochondrial beta barrel protein VDAC and I had success crystallizing intimin in them. David Bowie also has a JoVE article on the bicelle crystallization method. 3. Attempt crystallization using Lipidic Cubic Phases. This was the saving grace for my post-doc project. Neither detergent nor bicelle crystallization produced crystals that were of sufficient quality, but the crystals from LCP all diffracted to the 2.0 angstrom resolution range. If you're unfamiliar with the technique, there are several nice videos on JoVE describing it by Vadim Cherezov and Martin Caffrey. 4. Alter your construct. Small changes in the construct (ie: deletion or addition of 1-2 residues on the N- or C-terminus) often led to drastically different crystallization behavior of several OMPs in my hands. Cheers, Jim On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER <r.grinte...@research.gla.ac.uk<mailto:r.grinte...@research.gla.ac.uk>> wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures<http://www.emeraldbiostructures.com/> Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman....@gmail.com<mailto:fairman....@gmail.com> jfair...@embios.com<mailto:jfair...@embios.com>
