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Re: [ccp4bb] protein expression problem |
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CCP4bb navigationCCP4bb <-- 2008 <-- January 2008 <-- 22 January 2008Subject: Re: protein expression problem From: Juergen Bosch jbosch {- at -} U {- dot -} WASHINGTON {- dot -} EDU Date: 2008-01-22 things to check: - different media (LB. TB, auto) - when do you induce (mid log phase, or end log phase) - how long do you induce (before reaching stationary phase, going over night into starvation phase) - how much IPTG do you add - MBP purification, despite not being in the manual I noticed higher recovery rates when not following the protocol and using buffers ~ pH 8.5 - when you run your sizing how concentrated is your sample ? - do you have divalent ions in your buffer e.g. Mg or if you have them around, remove them by addition of EDTA or EGTA - glycerol in your sample buffer as chemical chaperone ? 10-20% would do it perhaps. So I would say, don't throw your construct away, unless you have tried all the other options first. Do you have a CD available, you could also check your sample if it's spaghetti or not. Good luck, Juergen Daniel Jin wrote: > Hi, > > I have been trying to express a rat protein in bacteria. The > MBP-fusion expressed at very high level (~ 40 mg/L) while the > GST-fusion and His-tag only gave inclusion bodies. The problem is that > all protein runs in the void volume on a size-exclusion column (s-200, > hepes, pH 7.4, 200 mM NaCl), no matter it is the intact MBP-fusion or > cleaved sample. There is no Cys on this protein so there is unlikely > any disulfide bond related problem. Anything I can do before I throw > away this construct and try insect or mammalian cells? Thanks. > > Best, > Chen > > ------------------------------------------------------------------------ > Never miss a thing. Make Yahoo your homepage. > -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch CCP4bb navigationCCP4bb <-- 2008 <-- January 2008 <-- 22 January 2008 |
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