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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: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU Date: 2008-01-22 Before adding detergent, be forewarned that the MPB in many fusions will not bind to an amylose column in the presence of most detergents, particularly maltoside detergents. It has been the bane to us so we have engineered MBP vectors with His tags to deal with this. What you might try, as suggested, the NDSBs or the addition of glycylglycine (to make 0.5-1.0M) to the growth media just before innoculation (don't worry about sterility with good antibiotic selection; autoclaving will just make a brown mess). The glycylglycine trick can reduce aggregation. Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: garavito@msu.edu **************************************************************** On Jan 22, 2008, at 2:23 AM, David Briggs wrote: > Hi Chen, > > 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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