pRSET/histag expression problems

[ijiwaru at wheel.dcn.davis.ca.us]

In article <Drobert2.3.0089E742 at utk.edu>, Drobert2 at utk.edu wrote:

> I have a problem with the expression of a membrane protein in E. coli using 
> the histag/pRSET system.  We are using the protease deficient strain
BL21lysE. 
>  The expression level is low (0.2 mg/liter culture) and much of the protein 
> does not contain a histag (apparently it is cleaved off during expression or 
> subsequent purification).  The protein does not accumulate in inlcusion
bodies 
> and apparently is inserted ok into membranes.  Has anyone else seen these 
> problems and could offer advice on how to improve expression/stability.

Several people have mentioned problems related to this on this group.  One
is the cloning of membrane proteins, the second is stability of clones in
BL21 and its derivatives.  I would recommend that you check the plasmid in
your cells to make sure it is intact, especially if you've been working
with the same culture for a while.  I would also suggest that you add some
glucose, 0.25%, to your culture media to keep the lacUV5 promoter shut
off.  Grow your culture to mid- to late-log phase and then rinse the cells
with media minus glucose and induce.  At least, you have a clone, we have
a couple people working in near-by labs who can't the their membrane
proteins cloned except in the opposite orientation with respect to the
promoter.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis