GST fusion: Solution and more questions

Pavel Sova ps44 at konichiwa.cc.columbia.edu
Mon Sep 26 15:03:46 EST 1994


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

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