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Site-Specific Cleavage of Fusion Proteins 219
Table 2
Conditions that can Alter Protease Specificity that are Compatible with Serine
Proteases
pH
Non-ionic
detergent
(%, v/v)
Ionic detergent
(%, w/v)
Chaotrope
(M)
NaCl (mM)
Temperature
(°C)
6.5 0.1 0.01 0.5 100 4
7.0 0.5 0.05 1 200 16
7.5 1 0.1 2 300 21–25
8.0 1.5 0.5 3 400 37
8.5 2 4 500
9.0
9.5
Note 19a, b Note 19c, d Note 19c, e Note 19c, f Note 19 (g) Note19h
12. Scale-up the successful reaction conditions 10-fold to provide a working preparation
of cleaved protein. Although individual reaction conditions and incubation
times will vary depending on those determined in steps 4–11, a generic reaction
protocol would be as follows: Mix 1 ml of fusion protein preparation (see step 2)
with 100 μl of protease dilution (see steps 3, 8–10); incubate at the required
temperature (see steps 4–11) for 1 h (if step 8 was followed) or 4–24 h (if steps
10 and 11 were followed); terminate the reaction by addition of 50 μl of 1 M
HCl or addition of appropriate protease inhibitors (see Table 3).
13. For notes on product purification and reaction cleanup, see Note 22.
Table 3
Common Protease Inhibitors
Inhibitor Protease class Molecular weight
Effective
concentration Notes
Aprotinin S 6500 10–250 μg/ml
Leupeptin hemisulphate S/C 475.6 1–100 μM 21
Phenylmethylsulfonyl S 174.2 0.1–1 mM
fluoride (PMSF)
Iodoacetic acid C 207.9 1–10 mM
Pefabloc® SC (AEBSF) S 239.7 0.1–2 mM
Pepstatin A A 685.9 0.5–1 μg/ml
Bestatin M (E) 344.8 1–150 μM
EDTA M 372.3 1–10 mM
E-64 C 357.4 1–10 μM
A, Aspartic; C, Cysteine; (E), Exoprotease; M, Metalloprotease; S, Serine.