Dr. R.J. Beynon
beynonrj at uxb.liv.ac.uk
Fri Oct 16 02:44:52 EST 1992
NANTEL at UNCVX1.BITNET wrote:
: 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
this is curious - can't really explain that - antipain and leupeptin
are pretty benign
that protease inhibitors are necessary since one of my fusion protein is
: chewed up from 95 to 80 kD during the purification.
: 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.
: Andre Nantel
: Biology Dept.
: University of North Carolina
And I'm a biochemist playig at being a molecular biologist :-)
Preventing proteolysis during proten purification can be a pain. Some silly
questions first -
Was the PMSF stock dissolved in water? - if so, it goes off with a half life
of minutes. Always make stock PMSF in dried solvents such as MeOH or PrOH.
PMSF only inhibits some serine proteases - there are another 3/4 classes
and some unknown class proteases lurking about. If you really want to eliminate
proteolysis, you have to be systematic, and start by making it happen (finding
conditions) and then trying to shut it down. There are dozens of inhibitors
that you could try.
You may find it helpful to have a look at the following:
Beynon, R.J. & Bond, J.S. Proteolytic Enzymes - A Practical Approach
Oxford University Press, (1989)
Harris, E.L.V. & Angal, S. Protein Purification - A Practical Approach
same (1989) - I wrte a section for that on preventing proteolysis
and Walker, J. Methods in Molecular Biology, Vol 3 (1988) I wrote a similar
chapter for that book p1-23.
This may seem like a bit of drum-beating, and it's not meant to be - :-)
I just happen to have worked with thesedelightful enzyme classes for
quite a while. Can't understand why everyone else hates them ;-)
Hell - why not dump your protein and work on the protease instead :-)
Lastly, we discovered some years back that sample preparation for SDS-PAGE can
cause some fragmentation too. Some of the degradation you see might be due to
There are two reasons for this
SDS unfolds substrates faster than proteases, so in a moment
of fleeting glory, the proteases go out with all guns blazing
and wipe out the substrate!
Excessive heating in SDS/reducing agent might be able to break up proteins
slightly as well.
We always TCA ppt proteins before dissolving in sample buffer.
Hope this helps
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