GST fusion: Solution and more questions
ps44 at namaste.cc.columbia.edu
Mon Sep 26 15:20:38 EST 1994
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
> Earle Stellwagen: Chromatography on Immobilized Reactive Dyes.
> Meth. Enz. 182, 343-357, 1990.
>/* 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
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
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 Sova e-mail:
Molecular Virology Laboratory ps44 at columbia.edu
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