Chen and David,
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: [EMAIL PROTECTED]
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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 <[EMAIL PROTECTED]> 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
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============================
David C. Briggs PhD
Father & Crystallographer
http://www.dbriggs.talktalk.net
AIM ID: dbassophile
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