View - ResearchGate

222 Charlton
3. The catalytic mechanism of cysteine proteases relies on the active site cysteine
thiol nucleophile. It is therefore vital to the activity of these enzymes that this
thiol be preserved, with the state of this functional group ensured by maintaining
a reducing environment. If this concentration of DTT causes reduction of labile
disulphide bonds in the target protein (determined by incubation of the protein
in cleavage buffer followed by analysis by a technique such as reversed phase
High Performance Liquid Chromatography (rp-HPLC)), then a milder redox pair
such as 3 mM reduced glutathione + 0.3 mM oxidized glutathione might be more
appropriate.
4. Cleavage prior to aspartate and glutamate can be improved >10-fold by cleavage
in 2 M KCl (manufacturer’s recommendation).
5. Chaotropes such as urea or guanidine–HCl are known to severely inhibit cleavage
by many proteases. The activity of these enzymes falls off sharply in the presence
of any chaotrope, often with undetectable activity in concentrations above 2 M
urea/1.5 M guanidine–HCl. Aside from decreased protease activity, the presence
of chaotropes can alter the specificity profile of the enzyme, potentially giving rise
to cleavage at unintended sites. It is therefore recommended that chaotropes be
avoided in the pilot cleavage experiments to avoid their unexpected interference.
6. Enterokinase and Factor Xa are inhibited by concentrations of salts (such as
NaCl) over 250 mM, as such it is recommended that the total concentration of all
salts not exceed this level in initial experiments. Imidazole is known to inhibit
these enzymes at concentrations over 50 mM. Although thrombin is generally
more salt and imidazole tolerant, with successful cleavage reported in 500 mM
NaCl and 500 mM imidazole (20), it is again advised that the total concentration
be kept below the stated thresholds if possible.
7. SDS, an anionic detergent, can inhibit cleavage at concentrations as low as
0.001%, but in practice, the effect of less than 0.01% should be negligible.
Although less information exists for enzyme inhibition by other charged detergents,
it is likely that they too cause a very similar loss of activity, and as such,
their presence in pilot cleavage experiments is not recommended.
8. Protease inhibitors may have been added at the cell lysis stage of protein purification.
9. Substrate concentration can have an effect on the rate of enzyme reactions.
Keep fusion protein concentrations as consistent as possible in pilot cleavage
experiments. Concentration of the fusion protein preparation can be performed
simultaneously with step 1.
10. One percent concentration of protease (relative to fusion protein) is the goal.
Use 1 unit of enzyme where the supplier defines a unit as having the ability to
cleave >90% of 100 μg. Some manufacturers may use a different unit definition,
in these circumstances; adjust the volume of protease added accordingly. For
example, if a particular manufacturer’s protease preparation defines one unit as
having the ability to cleave 50 μg of control protein, then double the volume of
protease added. Where both the mass (e.g., mg/ml) and the activity (units) of