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 <[log in to unmask]> 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.

-- 

============================

David C. Briggs PhD

Father & Crystallographer

http://www.dbriggs.talktalk.net

AIM ID: dbassophile

============================