View - ResearchGate

224 Charlton
may dictate. However, deviation outside the upper and lower limits specified
is unlikely to meet with a successful cleavage reaction outcome. The tertiary
structure of the protein can either inhibit protease action at the intended site
by sterically hindering accessibility, or promote incorrect internal cleavage by
exposing labile surface motifs. The non-exhaustive list in Table 2 or Table 4
suggests conditions that will mildly alter the protein structure without denaturing
the protease or protein product. Modification of multiple factors in concert may
be required for optimal outcomes. If the degree of correct cleavage is increased
by a factor, but not sufficiently so at any concentration/level, further cleavage
improvements may be made by holding this first factor constant at the level
that gave the best result and introduce a second variant factor and repeating the
optimization experiments.
a. Whilst not significantly altering the structure of the protein, the pH at
which the reaction is performed may be particularly useful for reducing
non-specific cleavage within the protein. As can be seen in Table 1, many
of the proteases recognize charge amino acid groupings; therefore, altering
the pH of the buffer can move toward or away from the pKa of the side
chains of ionizable amino acids. This can alter local charge environments
and can be sufficient to mask the secondary sites and prevent cleavage.
Similarly, varying the pH can cause localized charge modifications in the
protease active site that can shift the specificity of the enzyme enough to
discourage secondary cleavage.
b. Table 5 lists some common buffers that will be effective at the stated pH
points. Fifty millimolar solutions of each will provide sufficient buffering.
c. Inclusion of chaotropes or detergents will relax the structure of the protein.
These agents allow the normally buried hydrophobic residues of the
protein to become more solvent exposed by disrupting hydrogen bonding
and hydrophobic interactions. This can perturb the original structure of the
protein, providing greater exposure of the expected target cleavage site,
Table 5
Suitable Buffers at Given pH Ranges
6.5 7.0 7.5 8.0 8.5 9.0 9.5
citrate
MES
MOPS
MOPS
Tris-HCl Tris-HCl Tris-HCl
HEPES HEPES
Tricine Tricine Tricine
borate borate borate
CHES CHES