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218 Charlton
(see Notes 12 and 13). Terminate the negative control at 24 h. Store at –20°C
until all of the samples are ready to run on SDS-PAGE (see Note 14).
6. Analyze the time point samples and the negative control on SDS-PAGE.
7. If there is significant degradation of the target protein (see Note 15) go to step 8. If
there is incomplete cleavage (see Note 16), or no cleavage apparent where a positive
control was successful, go to step 10. If the cleavage was successful, go to step 12.
8. Incubation with a lower amount of protease may help to minimize (see Note 17)
internal cleavage of the target protein. Dilute the protease preparation to 0.005
and 0.0005 units/μl (or 5 and 0.5 ng/μl). To 2 × 20 μl of fusion protein from step
2, add 2 μl each of these protease dilutions. Incubate at approximately 21°C (see
Note 11)for1h(see Note 18). Terminate the reaction (see Note 13) and analyze.
If these reactions yield sufficient correctly cleaved target protein, go to step 12.
9. If overdegradation is still observed, reduce the concentration of protease further and
repeat the reaction. Further improvement in the yield of correct protein product may
be possible by altering the structural properties of the target protein (see step 11).
10. Increasing the concentration of protease may enable cleavage. Dilute the protease
stock to 0.25 and 0.5 units/μl (or μg/μl). Add 4 μl of each protease dilution to
40 μl of fusion protein from step 2. To another 40 μl of fusion protein, add 4 μl
of neat protease stock. Incubate at approximately 21°C (see Note 11). Remove
22 μl aliquots at 4 and 24 h. Terminate the reactions (see Note 13) and analyze
by SDS-PAGE. If these reactions yield sufficient correctly cleaved target protein,
go to step 12. If these protease concentrations remain unable to produce adequate
levels of correctly cleaved material, or if significant degradation of the target
protein is observed (see Note 15), go to step 11.
11. Alter reaction conditions (see Note 19).
a. Select one factor at one concentration/level from Table 2 to alter and
prepare fusion protein at 0.5 mg/ml in this variant cleavage buffer by
dialysis into the new system, or by adjustment of the original cleavage
buffer to include the new factor.
b. Dilute the protease preparation to 0.05 units/μl (or 0.05 μg/μl) in the
variant cleavage buffer. Keep protease preparations and stock on ice until
needed.
c. To 20 μl of the new fusion protein preparation (see step 11a), add 2 μl
of the new protease dilution (see step 11b) and incubate at the desired
temperature for either 1 h where reduction of internal cleavage is desired or
24 h where improvement of incomplete cleavage is the intended outcome.
Terminate the reaction at the appropriate time and analyze.
d. If the degree of correct cleavage is increased, but not sufficiently, further
improvement may be possible by altering the selected factor up or down,
and repeating steps 11a–c. If further improvement within one factor class
is not possible, hold this first factor constant at the level that gave the best
result and introduce a second variant factor, repeating steps 11a–c with
both factors.
e. See Note 20 for other avenues to achieve successful cleavage.