Bacterially Expressed Proteins: IPTG induction

[Randy Nonay]

Jay L. Rothstein (rothstei at lac.jci.tju.edu) wrote:
> We have cloned several pieces of genes into the invitrogen pTRCHis
> vector and have expressed them in DH10b, BL21 and similar bacteria.  The
> problem we have is that for some unknown reason we are unable to
> consistently induce these genes with IPTG.  Some days they induce fine
> other they do not (only background proteins can be seen)  We cannot find
> a correlation with  any feature of bacterial induction.  Does anyone
> have experience with this intermittant start/stop expression in a
> particular clone.  This can even occur in situations where one induction
> works but the next day it doesn't with the same bacteria (taken froma
> frozen stock or from a plate).  We have also altered IPTG stocks and
> levels but it does not appear to be cause by the IPTG.  Also the same
> vector in DH10B or XL1 blue or BL21 cells behave the same way in that we
> will intermittantly see no IPTG induction. While we CAN get induction
> this inconsistency is annoying and wasteful.  Any suggestions?  Has
> anyone experineced this probelm with bacterial expression vectors?
> -- 
> __________________________________________________________

> Jay L. Rothstein, Ph.D.   
> Thomas Jefferson University 
> Departments  Micro/Immuno/Otolaryngology
> Kimmel Cancer Institute, Philadelphia, PA 19107 
> 215-503-4623:::Fax: 215-923-3473
> rothstei at lac.jci.tju.edu
> ___________________________________________________________

   One thing to avoid (particularly if your expressed protein is toxic to
the host) is over-growing the culture. If you go too far, your yeild will
drop substantially. With some strains I was working on, if I let them grow
overnight they were usually worthless. I always tried to grow them to only
about .3-.6 OD A600 before inducing. Got great results.

 Randy Nonay
 Dept. of Chemistry, University of Alberta