inclusion bodies

[Ted M.]

In article <tedm-2407952303590001 at cisco-ts5-line1.uoregon.edu>,
tedm at darkwing.uoregon.edu (Ted M.) wrote:

> 
> 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

A number of people have written regarding the ref to chaparone
coexpression for recombinant proteins... I found the ref but I should
remember to read my current contents more carefully,  The ref regards
coexpression of secB, DnaK & J and groES/groEL with GCSF, not GMCSF. Their
positive result was with DnaK & J and periplasmic directed leader. The ref
is 
   
   Perez-Perez, J.,et al Biochem. Biophys. Research Comm. vol 210, #2, p 524

This work is along the same lines as several projects from industry, which
are not published. The authors ref two papers on similar attempts. Good
luck all.

                                                 Ted Michelini
>                                                Institute of Molecular Biology
>                                                University of Oregon
>                                                tedm at darkwing.uoregon.edu