I would definitely try gelfiltration (how do you get rid of the cleaved tag anyway, sample buffer exchange?) but especially ion exchange. A homogeneous sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in ion exchange. Beyond that i would make some point mutations on surface residues and focus on those (ie try the surface entropy method).
Good luck, Bert
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From: CCP4 bulletin board [[log in to unmask]] On Behalf Of eswar reddy [[log in to unmask]]
Sent: Friday, August 26, 2011 6:56 AM
To: [log in to unmask]
Subject: [ccp4bb] Crystals with Organic solvents
Dear All
I was working on a Human protein and expression and solubility is good in E.coli and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli shaped and i tried seeding, but it is not successful, and even i tried with additive screen but the result is the same .... is there is any idea to increase the size and shape of my protein crystals.
Any suggestions will be helpful for me
Thanks in Advance
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