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Site-Specific Cleavage of Fusion Proteins 223
the protease preparation are supplied, use the activity measure to determine the
amount of protease to use.
11. Room temperature is acceptable if constant and within 20–25°C.
12. A reducing SDS-PAGE will show if the protease has cleaved protein product
internally. An adventitiously cut protein product may appear intact on a nonreducing
gel if held together by disulphide bonds.
13. The constituents of SDS–PAGE loading buffer, particularly the high concentration
of SDS, will very effectively terminate all protease activity. If not
using SDS–PAGE analysis, the reaction may be terminated by acidification, for
example, add 3–5 μl 1 M HCl, or by the addition of a protease inhibitors against
the added enzyme, as listed in Table 3.
14. Select a SDS-PAGE system that will allow separation in the range between the
size of the full-size fusion protein and the successfully cleaved target protein.
Bear in mind that there may be smaller fragments present if the protein has been
overdegraded.
15. Degradation of the protein product is indicated by a decreased abundance of
material with the correct mass and the appearance of smaller products that were
not present in the initial preparation or in the negative control sample. These
effects will usually become more pronounced over the time course. In some
cases, the fusion partner may be visible by SDS–PAGE. Ensure its presence is
not mistaken for an internal cleavage fragment. If degradation is observed in
the protease negative control, there may be contamination of the fusion protein
sample by other proteases. Consider further purification.
16. In many cases, incomplete cleavage is preferable to overdegradation as intact
fusion protein is more readily separated from the correct protein product than
that protein will be from internal cleavage fragments, see Note 22.
17. Where internal cleavage of the protein has occurred, it is unlikely to be completely
avoided. If the presence of these breakdown fragments or the associated yield
losses cannot be tolerated, consider using another protease system.
18. It is assumed that the protein was overdegraded at the 1-h point in the initial
time course. In most cases, a lower protease concentration will not change the
cleavage profile (the products that are generated) substantially, but will instead
increase the time taken to achieve the same profile. Performing the reaction
at a lower protease concentration can be thought of as somewhat analogous to
expanding the time taken to create the reaction products. Thus, it is possible to
collect the reaction products at time points that would have been impractical to
capture at the initial reaction ratio, such as those that formed in the first few
minutes or seconds of the reaction.
19. Alteration of reaction buffer conditions may promote correct specificity. Table 2
(see Subheading 3.2., serine proteases) or Table 4 (see Subheading 3.3., cysteine
proteases) suggest a range of potential reaction condition variations in which the
specificity of the protease may be sufficiently altered to enable hydrolysis at the
intended site. The concentration/level value ranges provided are intended as a
guide only, with any amount within those ranges acceptable as circumstances