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220 Charlton
3.3. Cleavage of Fusion Proteins with Cysteine Proteases
1. If the fusion protein sample contains urea or guanidine (see Note 5), ionic
detergents >0.01% (see Note 6), Zn ++ >5 mM (see Note 23) or known protease
inhibitors (see Note 8), dialyze into cleavage buffer.
2. Concentrate or dilute the fusion protein preparation to approximately 0.5 mg/ml
(see Note 9).
3. Dilute the protease preparation to 0.05 units/μl (or 0.05 μg/μl) in cleavage
buffer (see Note 10). Keep protease preparations and stock on ice until
needed.
4. Set up a pilot cleavage by mixing 100 μl of fusion protein (50 μg at 0.5 μg/μl,
see step 2) with 10 μl of protease dilution (see step 3). Prepare a negative control
reaction by adding 2 μl of cleavage buffer to 20 μl of fusion protein preparation.
Incubate these reactions at 4°C (see Note 24). If a positive cleavage control was
supplied, prepare this reaction according to the manufacturer’s directions.
5. Terminate the reactions after 24 h by adding 22 μl of 2× reducing SDS-PAGE
loading buffer (see Notes 12 and 13). Store at –20°C until the samples are ready
to run on SDS-PAGE (see Note 14).
6. Analyze the time point samples and the control(s) on SDS-PAGE.
7. If there is significant degradation of the target protein (see Note 25) 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. Carefully analyze the negative (no protease) control (see Note 26); if degradation
is observed in this reaction, consider expression in a host protease-deficient
bacterial strain such as Escherichia coli BL21(DE3). The inclusion of protease
inhibitors that do not affect cysteine proteases may also be beneficial, see
Table 3. Return to step 4 with inhibitor inclusions or new host strain. Where
the degradation is observed to be attributable to the viral protease, continue to
step 9.
9. 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 4°C for 24 h. Terminate
the reaction (see Note 13) and analyze by SDS-PAGE. If these reactions yield
sufficient correctly cleaved target protein, go to step 12. Otherwise continue to
step 11.
10. Increasing the concentration of protease may enable cleavage. Dilute the protease
preparation 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 the reactions at 4°C for 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, go to
step 11.