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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: David Briggs drdavidcbriggs {- at -} GMAIL {- dot -} COM Date: 2008-01-22 You could try adding some detergent or other solubilising agent (eg NDSBs) to your buffer. Have you tried other pHs? If you are sat near to or on the pI of your protein, it will be at its least soluble and more likely to aggregate. I've had protein behave like yours at pH 7.5 but behave perfectly (i.e. monodisperse) at pH 5.5. As you can get you protein in inclusion bodies, have you considered doing an inclusion body prep (using 'bugbuster' or something similar) and then trying some refolding protocols? Jungbauer A, Kaar W. Current status of technical protein refolding.J Biotechnol. 2007 Feb 20;128(3):587-96. Some people have had success with SUMO tags as well. HTH, Cheers, David On 22/01/2008, Daniel Jin > > 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. > > -- ============================ David C. Briggs PhD Father & Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile ============================ CCP4bb navigationCCP4bb <-- 2008 <-- January 2008 <-- 22 January 2008 |
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