E. coli proteolysis

[Bob Steinberg]

Randy Willis wrote:
> 
> Rob wrote:
> > We've been trying to express a pro-hormone as a GST fusion in E. coli
> > (DH5-alpha) but seem to be having problems with proteolysis - we think this
> > may be occuring at double basic sites but are not completely sure.
> >
> > Has anyone
> > a) had similar problems
> > b) been able to solve the problem by using e.g. JM105s or BL21s
> > c) tried inhibiting endogenous coli proteases (i.e. whilst they are
> > growing) by adding protease inhibitors to the broth (will they get into the
> > cells ?)
> >
> 
> Rob and Shi Tao,
> 
> Only with one of my proteins, expressed on a pET vector and in BL21(DE3)
> cells, had problems with proteolysis.  I was able to limit some of the
> clipping through an aggressive bath of protease inhibitors, but some
> clipping occured in the cells during synthesis.  I got around this
> problem by growing the culture at lower temps and inducing later in log
> phase.
> 
> Start the culture at 37C at OD600~0.05
> At OD~0.2, change temp to 30C
> At OD~0.4, change temp to 25C
> At OD~0.6, change temp to 20C
> At OD~0.8, change temp to 15C
> At OD~1.2, induce and allow to grow at 15C for 8-36h
> 
> My growths were all in minimal media (we're an NMR lab), but this
> protocol ended up giving me the best yields and highest final OD's that
> we have ever experienced in my lab (final OD >3).
> 
> Good luck,
> 
> Randall C Willis, Researcher
> Biochemistry, Hosp for Sick Children
> 3522-555 University Ave
> Toronto, ON
> M5G 1X8  CANADA
> 
> 416-813-5933 (ph)  -5022(fax)
> 
> willis at gandalf.psf.sickkids.on.ca

What sort of device do you use to incubate your cultures below room
temperature??