inclusion bodies

Ted M. tedm at darkwing.uoregon.edu
Tue Jul 25 00:49:00 EST 1995


In article <ifag.9.0 at po.uni-stuttgart.de>, ifag at po.uni-stuttgart.de
(Volker Seibert) wrote:

> Hi everyone!
> I want to express a protein from a gram (+) strain (Rhodococcus erythropolis 
> 1CP) in E. coli BL 21 (DE3)pLysS. I am using pET11a and pRSET6a vectors. 
> Unfortunately, I get most of the protein in inclusion bodies. I have tried 
> several conditions including different temperatures (25, 30 and 37øC), 
> additives (betaine, sorbitol) and media (M9 and dYT). Renaturation from 
> inclusion bodies was not successful. Can anyone help me?
> Thanks in advance.
> Volker

Volker,
   This is not a trivial problem; the biotech industry has spent millions
trying to make proteins work in coli. You have tried some of the right
things; temperature can be crucial with some protein requiring ~20°C to be
soluble (while growth is nil) You are using a very strong promoter system,
it is possible that titrating the IPTG, or using lactose could give you
some soluble protein.In my experience with huIL7, the less IPTG, the more
soluble protein. Others have had success using periplasmic directing
leaders in coli, including ompA and pho. Invitrogen claims thioredoxin
fusion proteins are more soluble, but this is very case specific. One
possibility is the use of heat shock/chaparone proteins to enhance correct
folding of recombinant proteins. This can be a simple as a 5min shock at
42°C followed by 15 min at 40°C and then lower the temp to 25°C for IPTG
induction! Or you can coexpress GroEL & S, as was tried recently with
Gm-CSF. It can all get quite complex! 
   What is essential in these explorations is a facile assay for
solubility and a knowledge of your protein. Are there lats of cystein
bridges? Tried disulfide isomerase in the refolding mix? etc etc. Good
luck.
                                                  Ted Michelini
                                                  Institute of Molecular Biology
                                                  University of Oregon
                                                  tedm at darkwing.uoregon.edu



More information about the Methods mailing list