GST fusion: Solution and more questions

[Pavel Sova]

In article ,  <> wrote:
>Kim Hak-Sung (hskim at sorak.kaist.ac.kr) wrote:
>
>: Recently, I am trying to purify an enzyme which needs NAD+ as a cofactor.
>: I heard that there're some affinity columns which can be used for 
>: this purpose.
>
>Cibacron Blue columns are used to bind proteins which have NAD as cofactor.
>There are lots of literature about this method (in about any book on protein
>purification there is a chapter dedicated to this topic), but for a start
>try
>	Earle Stellwagen: Chromatography on Immobilized Reactive Dyes.
>	Meth. Enz. 182, 343-357, 1990.
>
>--Cornelius.
>
>--
>/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */
>/* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3795 */
>/* "People are DNA's way of making more DNA." (Edward O. Wilson, 1975)    */

GST-fusion: solution and more questions

Hi bionetters,

I posted here couple of months ago a question about isolation of 
GST-fusion protein. My problem was that 23-kD protein hooked up behind 
GST wouldn't bind to a G-Sepharose, after I was able to solubilize it 
from inclusion bodies (which contained 100% of my fusion protein) by 1.25% 
Sarcosyl in Tri-ethanolamine buffer. The problem was that I 
omitted final step before binding it to G-Sepharose, that is bringing up 
the solubilized protein to 2% Triton X-100 concentration. This step, at 
least I think, should sequester the SDS-like compound, Sarcosyl, to micelles 
and thus remove it from protein which is in my case, probably denatured by 
Sarcosyl.

After adding the TX-100 step, I was able to get some binding to 
G-Sepharose, but most of the fusion protein (say 85%) remained in 
solution after spinning down the Sepharose beads. The same I can observe 
with halves of my protein's open reading frame fused to GST. Better 
results I get if I solubilize inclusion bodies in 0.5% Sarcosyl solution, 
suggesting that incomplete removal from protein surface is at play.

I am wondering how to increase ability of fusion proteins to bind to a 
G-Sepharose. One protocol (article by Frankel et al., PNAS 
88:1192-6,1991) suggests to use detergent octyl-glucoside to sequester 
sarcosyl in micelles; they used sarcosyl in 1.5% concentration to disolve 
inclusion bodies. They don't write what concentration of octyl-glucoside 
they used, though. Do you anybody have any idea what concentration 
of octyl-glucoside should be used?              ^^^^^^^^^^^^^^^^^^           
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Also, in my next pursuit I am going to try solubilization in SDS and then 
removal of it either by precipitation with KCl (Huang et al. BioTechniques 
15:989, 1993), although one try of this method did not give me good 
result, and then maybe another attempt to remove SDS (or perhaps also 
Sarcosyl in parallel experiment) with ion-exchange resin AG 11A8 from 
Bio-Rad. If you anybody have experience or ideas with this, please let me 
know or post it here, because I know that GST-fusion proteins are used very 
often and many people have all kinds of troubles with them.

Thanks in advance for any input
Pavel

 -----------------------------------------------------
 Pavel Sova                          e-mail:
 Molecular Virology Laboratory       ps44 at columbia.edu
 Columbia University
 New York                            
 -----------------------------------------------------