inclusion bodies

Bernard Heymann bheymann at bragg.bio.purdue.edu
Wed Apr 20 22:30:23 EST 1994


In article <199404201821.LAA14280 at net.bio.net>,
N490047 at UNIVSCVM.CSD.SCAROLINA.EDU (Tom MacAllister) wrote:

> 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.
> 
>                                                                      Tom
> 
> TOM MACALLISTER
> N490047 at UNIVSCVM

I think the common wisdom on inclusion bodies of overproduced proteins is
that the protein production process is too fast to allow proper folding of
the protein. One of the approaches to deal with inclusion bodies is to try
and solubilize it with detergents, which is sometimes in itself difficult.
However, when you know that the protein in the inclusion bodies is
inactive, the only viable approach is to change the growth conditions. The
most commonly tried way is to just grow the cells at low temperature, of
course with a time penalty. I know of one person who needs to grow her
cultures at 20¡C for three to four days to get about 20 mg protein per
liter of culture. Other possibilities to try are growing it under oxygen
limitation (i.e. slower shaking), or stressing the cells into producing
chaperone-like proteins. The one example I know of in the latter category
involves the addition of ethanol to the growth medium (I think about 1 %).
All of these measures of course need to be tailored to your particular
protein. So happy trial-and-error ;-)


-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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