Protease inhibitors

rpgrant at vax.path.ox.ac.uk rpgrant at vax.path.ox.ac.uk
Wed Oct 14 07:20:21 EST 1992


In article <9210131601.AA11710 at net.bio.net>, NANTEL at UNCVX1.BITNET writes:
> 
> Greetings Netters!!
> 
> I'm fishing for information concerning the use of protease inhibitors during
> the purification of MBP-fusion proteins from E.coli. PMSF doesn't seem to
> have any effects on the quality of my protein preps. Furthermore, when I
> use antipain + leupeptin the efficiency of the purification step through
> the amylose column is greatly reduced (7-10 fold lower yields). I feel
> that protease inhibitors are necessary since one of my fusion protein is
> chewed up from 95 to 80 kD during the purification.
A couple of suggestions;

i)  During the lysis procedure, watch the sonication!!!  This has been a 
source of grief to others.  Excessive sonication does appear to result in 
degradation.  I find 4 x 15 second blasts, with 30s intervals, works well for 
25ml resuspended bugs.  A colleague uses 3 x 30s, with 30s intervals, for 40ml 
solutions.  Keep it on ice ALL the time.

ii) Do you know at what stage degradation occurs?  The relevant chapter in 
"Current Protocols in Molecular Biology" (the Red Book) suggests use of 
* protease-deficient *strains of E.coli, such as lon, htpR, DnaJ for pMALc2 
fusions, depP and tsp for pMALp2 fusions (and probably c2, as well), 
and ompT for both.  

> I am faily new to the world of protein purification (yes, another molecular
> biologist doing biochemistry without a licence) and any suggestions would
> be most welcome.

So who needs a licence? 

> Andre Nantel
> Biology Dept.
> University of North Carolina


Richard Grant
Sir William Dunn School of Pathology
Oxford.



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