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Site-Specific Cleavage of Fusion Proteins 221
Table 4
Conditions that can Alter Protease Specificity that are Compatible with
Cysteine Proteases
pH Non-ionic detergent (%, v/v) NaCl (mM) Temperature (°C)
6.5 0.1 200 4
7.0 0.5 300 16
7.5 1 400 21-25
8.0 1.5 500 34
8.5 2 800
9.0 1000
9.5
Note 19a, b Note 19c, d Notes 19g and 27 Note 19 h
11. Alter reaction conditions (see Note 19):
a. Select one factor at one concentration/level from Table 4 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 24 h. Terminate the reaction and analyze.
d. Increase or decrease the factor iteratively by repeating steps 11a–c until
successful cleavage is obtained.
e. See Note 20 for other avenues to achieve successful cleavage
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, 9–11); incubate at the required
temperature (see step 4 or 11) for 24 h. 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.
4. Notes
1. Other buffers for this pH are acceptable, such as N-(2-hydroxyethyl) piperazine-
N´-(2-ethanesulfonic acid) (HEPES).
2. The action of these proteases is enhanced by inclusion of low levels of NaCl and
trace CaCl 2 (19).