E. coli proteolysis

[Randy Willis]

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