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Site-Specific Cleavage of Fusion Proteins 225
potentially shifting the equilibrium of the cleavage reaction away from the
secondary site and toward the primary. Although Note 5 cautions against
the use of chaotropes, successful cleavage is indeed possible under these
conditions, with successful cleavage reported by Enterokinase in 2 M urea
(21), and Genenase I in 2.5 M urea (22,23). However, the activity of the
proteases will most likely be significantly decreased, requiring a higher
concentration of enzyme. The inclusion of chaotropes will most likely
require a concurrent re-examination of the amount of enzyme used, as in
step 10.
d. Examples of common non-ionic detergents are Tween-20 and Triton X-
100.
e. An example of a common ionic detergent is SDS. Ionic detergents should
be used sparingly, as they are powerful protein denaturants.
f. The most commonly used chaotropes are urea and guanidine–HCl. The
concentrations given in Tables 2 and 4 are based on urea; if guanidine-HCl
is used instead, decrease these values by 25%.
g. The inclusion of NaCl can relax protein structure by reducing the stabilizing
effect of salt bridges. The inclusion of NaCl alone is unlikely to
alter the initial cleavage profile, but can synergistically act with the other
suggested factors to improve the overall specificity of the protease.
h. Aside from directly contributing to the rate of the protease reaction in
a manner much similar to alteration in the enzyme : substrate ratio,
the temperature of the incubation can also have an effect on protein
structure. As decreased temperatures weaken hydrophobic interactions and
strengthen hydrogen bonds and vice versa, there exists the potential to
alter the cleavage profile of the system by simply altering the incubation
temperature (author’s personal observations).
20. If successful cleavage is still not obtained but the use of the selected protease
is still desired, consider the insertion of a tetra- to hexa-peptide spacer sequence
N terminal to the protease recognition sequence. The inclusion of a flexible
spacer peptide sequence can allow greater access to the intended cleavage site by
minimizing steric inhibition by the fusion partner. The steric inhibition effect can
be particularly prevalent when dealing with small, largely unstructured peptide
fusions that are able to fold back onto the protein structure, occluding the cleavage
site (author’s personal observations). For serine proteases, sequences such as S 3 G
(24), SG 4 A (25) and SG 5 (26) have been used successfully for this purpose. As
viral proteases tolerate little deviation from the wild-type recognition sequence,
an upstream spacer derived from their wild-type polyprotein sequences may be
more useful than an artificial polypeptide at reducing steric interference. In the
case of TEV, such a sequence is DYDIPTT (27), and for HRV, a similar candidate
is KMQITDS (28). Return to Subheading 3.2., step 1 or Subheading 3.3., step
1 with the new fusion construct
21. Leupeptin may also inhibit viral cysteine proteases at concentrations over 100
μM (29).