detergent removal and protein precipitation

Hi all,

I am purifying a diffusive signaling ligand from E. coli BL21(DE3)
lysate. According to the literature, its monomer is hardly soluble in
aqueous solvent (my result is consistent). Crystal structure predicts it
to be soluble upon disulphide bond-involved dimerization. I would like
to seek your advice on the following questions or to my work in general:

1. I plan to use a mild detergent to solubilize the monomer, subject it
under disulphide-bond promoting conditions, and remove the detergent
afterwards. My lab has SDS, Triton-X-100, NP-40 and Tween-20 only. Will
Tween-20 suit my purpose?

2. How to remove Tween-20 from protein sample? I am thinking about using
acetone (in excess volume) precipitation because tween-20 is soluble in
acetone. Will this work?

3. What's the usual effect of protein precipitation on protein? What's
the difference among precipitation reagents?

4. I am considering to use a E. coli strain that allows disulphide-bond
formation in cytoplasm. Is the Origami(TM) series from Novagen my only
choice? Can you share your experience in these strains or others ?


Your insights and kind sharing are heartedly appreciated.

Martin


---

"Martin" wrote:



SDS is everything but mild, for most proteins it's strongly denaturing.
The non-ionic detergents are milder. You may also try
cetyltrimethylammonium bromide (CTAB), a positively charged detergent,
highly solubilising but surprisingly mild. Also relatively cheap.

Aceton precipitation is a classical method for protein purification, but
acetone is denaturing on proteins, especially at higher temperatures.
Use the aceton very cold (-20 degrees or less) and add it slowly to the
ice-cold sample as mixing acetone and water produces heat. Spin the
protein precipitate down (at -10 degrees) and lyophilise the pellet to
remove the acetone. Only when all acetone is removed re-dissolve in a
buffer of your choice. For small volumes, methanol/chloroform
precipitation (Wessel & Fluegge) allows better recovery.

Alternatively, use BioBeads (from BioRad) to bind the detergent by
hydrophobic interaction. The protein stays in solution. The following
review (available on the net) should direct you to detailed description
of the method:

@article{Rig-98,
AUTHOR= {J.L. Rigaud},
TITLE= {Membrane proteins: functional and structural studies
using reconstituted proteoliposomes and 2-{D} crystals},
YEAR= {2002},
JOURNAL= {Braz. J. Med. Biol. Res.},
PAGES= {753-766},
VOLUME= {35},
LANGUAGE= {engl}
}

Pierce offers small disposable columns of a similar material for
detergent removal from proteins.


That depends on the protein. Some proteins are difficult to redissolve,
others survive the procedure quite well. Impossible to predict.

[jsrsol]

i wasn't aware of this information of detergent removal .
thanks Martin for such a great information.

[jsrsol]

i wasn't aware of this information of detergent removal .
thanks Martin for such a great information.