IUBio

How to avoid inclusion bodies ?

Joe Mack mack at ncifcrf.gov
Tue May 23 09:18:57 EST 1995


In article <3pqbq5$lu1 at n.ruf.uni-freiburg.de> vonrhein at bio5.chemie.uni-freiburg.de (Clemens Vonrhein) writes:
>The subject says it all: I want to avoid the expression of a protein
>as inclusion bodies.

My experience has been that once you have inclusion bodies, you're faced
with a lot of work to get it soluble and active, and with low probability
of success. I would suggest another project (really). I don't think
many (or maybe any) proteins that have been refolded have crystallised
for instance (some indication of their lack of homogeneity). I have
tried different clones (chopping off a few residues at the ends) which
succeeded in making a soluble expression system, with a protein of
40kd, but no success with a bigger protein. Changing from e.coli to
yeast fixed one system I know of.

	Joe Mack
	mack at ncifcrf.gov


>
>As far as I read the literature, there are some rules of thumb:
>
>- lower temperature (30, 25, 20, 15 degrees Celsius ?)
>- low IPTG concentration
>- rich medium ( terific broth, ...)
>- high OD and short incubation times (OD=1.0-2.0 and t=1-2h)
>- adding needed cofactors (FAD, FMN, ...)
>
>Are these rules true in general ? Is there a good and recent review ?
>What are your own experiences ?
>
>Is it worth trying to get soluble protein ? Or is it better to get a 
>lot of protein as inclusion bodies and try to refold it ?
>
>BTW, I use a pET-22/BL21 system.
>
>Any hints/answers/tips .. highly apreciated.
>
>Thanks
>
>Clemens
>---
>
>***************************************************************************
>* Clemens Vonrhein   email vonrhein at bio5.chemie.uni-freiburg.de           *
>*                    WWW   http://bio5.chemie.uni-freiburg.de/~vonrhein/  *
>*                                                                         *
>*         Institut f. Org. Chemie u. Biochemie                            *
>*         Albertstr.21                                                    *
>* D-79104 Freiburg i.Br. (Germany)                                        *
>*  Tel.: 761/203-6061   FAX: 761/203-5987                                 *
>***************************************************************************





More information about the Proteins mailing list

Send comments to us at biosci-help [At] net.bio.net