GST fusion: Solution and more questions

Kevin Pumiglia pumiglk at aa.wl.com
Tue Sep 27 08:20:42 EST 1994


In article <3679f2$8ih at apakabar.cc.columbia.edu>,
ps44 at konichiwa.cc.columbia.edu (Pavel Sova) wrote:

> 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 the 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 
> adding Triton X-100 to 2% concentration to solubilized protein. This 
> step, I suppose, 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 of Sarcosyl 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                            
>  -----------------------------------------------------

An article by Frangioni and Neel in Analytical Biochemistry (210:179, 1993)
gives an overview of Sarkosyl lysis under different conditions.  I have
utilized there procedures outlined in this report for difficult to
solubilize fusions and found that they work resonably well.  In short
several determining factors are the removal of Sakosyl by mixed micelles. 
The triton-X concentration is usually 2% for a lysis in 1.5% Sarkosyl, but
for stubborn proteins may be increased and improved by increasing up to 4%
TX-100.  The absence of divalent cations will improve the removal of
Sarkosyl; gentle non-denaturing lysis, and  presence of DTT may all improve
binding to the Glutathione-Sepharose.  Good luck!



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