Inhibition of uncharacterized protease(s) in growth medium

Robert Preston rapr at MED.PITT.EDU
Fri Jun 18 14:20:55 EST 1993


> In article <01GZF6Q5ME2Q8WWCU0 at INVERMAY.CRI.NZ>, VANNESTEJ%RUASV1 at ruakura.cri.nz writes:
> > 
> > 	Hello,
> > 
> > 	I am working on a gene which product is inhibitory to some 
> > Enterobacteriaceae (gram negative close to E. coli).  This inhibitory compound 
> > is sensitive to proteases.  The problem is that the strains I am using to 
> > detect the inhibitory activity do produce some proteases.  I am thus wondering 
> > if I could increase the sensitivity of my assay by getting rid of the 
> > extracellular proteases.  This means I would need a protease inhibitor that 
> > can be added to the growth medium without harming/killing the bacteria.
> > 
> > 	Any pointer appreciated
> > 
There was a related problem with yeast alpha-factor assays.  One way to
reduce proteolysis of the factor (or, anyway, increase assay sensitivity)
was to reduce the pH of the assay plates.  A much more elegant way came
with the discovery of mutant indicator strains that lack the protease
that normally cleaves the alpha factor.  You could screen for an
analogous mutant to use in your inhibitor assay.    ......Rob

Robert A. Preston
rapr at med.pitt.edu




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