inclusion bodies

Peter Gegenheimer peterg at rnaworld.bio.ukans.edu
Fri Apr 22 03:55:05 EST 1994


In <199404201821.LAA14280 at net.bio.net>, N490047 at UNIVSCVM.CSD.SCAROLINA.EDU (Tom MacAllister) writes:
>Dear netters:
>     We have a construct that produces major amounts of our protein of interest
>, but it is insoluble.  We have achieved resolublization, but have not
>recovered any activity.  The properly folded protein is active (no mutations)
>because we can assay it in the sup.  The problem is that ~99% is in the pellet.
>Facts: 1. It resolublizes in 2M GuCl or 4M urea.  2.  It stays soluble in 1M
>GuCl when dialyzed.  3.  We believe that there is at least 1 disulfide bond.
>4.  It is mostly insoluble in higher salt (150 - 500mM) in 1M GuCl.  5.  Dilu-
>tion doesn't work any better than dialysis.  6.  We have been using 50mM Tris,
>pH7.9 at 4C.  7.  10% glyerol doesn't help.   Does anyone have any experience
>with this or know anyone that would be good to talk with?  Thank you.

Couple of things to try: step-wise dialysis vs. sequentially lower [GuHCl] or 
[urea] in 3 or 4 steps; try Urea vs GuHCl; include substrate in all dialyses 
(probably the most important thing you can do!); systematically vary pH and temp
- sometimes higher temp may work better. All of the above were necessary for 
refolding of chloroplast ATP synthase subunits (Chen et al, FEBS Lett. 298,
69-73, 1992). Can also vary glycerol (0..25%) and/or  PEG (0..8%).

o-------------------------------------------------------------------------------o
|  Peter Gegenheimer                          |  pgegen at kuhub.cc.ukans.edu      |
|  Departments of Biochemistry and of Botany  |  voice: 913-864-3939            |
|  University of Kansas                       |  FAX  : 913-864-5321            |
|  2045 Haworth Hall                          | "The sleep of reason produces   |
|  Lawrence  KS  66045-2106                   |  monsters."              Goya   |
o_____________________________________________|_________________________________o




More information about the Methods mailing list