How to prevent proteolytic degradation

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Tue Nov 7 00:51:16 EST 2000


In article <8u6qr1$cg4$1 at pegasus.csx.cam.ac.uk>,
  "Choe Woo-seok" <wsc22 at cam.ac.uk> wrote:
> Dear all,
> We have the inclusion body protein expressed from E. coli HMS174(DE3).
> When the IB protein is dissolved in 50 mM Tris, 50 mM DTT, 8M Urea, pH
8.0,
> the proteolytic
> degradation of target protein (Mw = 65 kd, with 6xHis tag) started.
We
> repeatedly saw four
> four major degradation products on the 12% gel, and the degradation
rate
> looks quite fast.

There are a couple of major types of endoproteases, named after their
active centre and each inhibited by (more or less) specific compounds:
Serine (inhibited by PMSF, aprotinin, leupeptin, TLCK, TPCK), Cysteine
(chymostatin, E64, leupeptin, TLCK, TPCK), aspartic acid (pepstatin) and
metalloproteinases (EDTA, EGTA, phosphoramidon). I have also found it
usefull to include 6-aminocaproic acid, even though this substance is
said to be specific for the proteases of the blood clotting cascade (it
is not).

There are also amino- (bestain) and carboxypeptidases, which clip of
amino acids or short peptides from the end of a protein.

Serine proteases are probably the most frequently encountered, and PMSF
is the cheapest inhibitor. Two things need to be considered with this
substance: It is unstable in water (will be inactive after about 20 min)
and needs to be added to the buffer immediately before use from a stock
solution in aprotic solvent (like DMSO). It is also very toxic (blocks
acetylcholine esterase, which is also serine dependent). This problem is
amplified in DMSO solutions, as this solvent makes the skin permeable
for xenobiotics. There are some compounds available that act like PMSF
but are less toxic (AEBSF, PTSF). Unfortunately, they are expensive.

When I need heavy protection against proteases, such as during cell
homogenisation, I use a protease inhibitor mix (1 mM EDTA, 5 mM
6-aminocaproic acid, 0.1 mM Benzamidine, and, from a 1000-fold stock
solution in DMSO 1 uM each of PMSF, leupeptin, pepstatin, E64.
Aprotinine can sometimes also help. Note that some of these compounds
are very expensive, it pays to find out which are needed and which are
not.

Boehringer (now Roche) has inhibitor coctail tablets, which are useful
if you can aford them.


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