In addition I would try expressing very late and short. Growing up your cells to the maximum OD600 and then inducing for as short as you can at lower temperature e.g. 16-20 ˚C Your protein might be degraded while expressed.
Jürgen On Aug 21, 2012, at 4:28 PM, Das, Debanu wrote: Hi Theresa, The deletion probably led to a folding problem, making it susceptible to residual proteases. You might try the following: -Lysis and purification in the presence of 1mM EDTA, which can help to neutralize proteases in addition to the protease inhibitor, without significant leaching of metal in a metal-affinity column (assuming your protein does not have natively-bound metal at an active site required for stability) -Try cocktails of different protease inhibitors -Denature the target completely with urea of GuHCl and then try refolding after purification efforts -In case your target of interest is from a thermostable organism and expressed in E. coli, you can try heating the sample to denature contaminating proteins/proteases. Best, Debanu. -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa Hsu Sent: Tuesday, August 21, 2012 1:15 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Purify non-stable protein Dear all I made deletion mutation of a stretch of 20 amino acids on my protein. I can purify and crystallize wild type protein but not the mutated. Mass spec on gel separated protein shows degradation of mutant losing about another 150 amino acids. Is there any way of purifying this non-stable protein? I know nature has designed proteins to be stable. All steps are done at 4 C and protease inhibitor added during cell lysis for both proteins. Thank you. ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
