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216 Charlton
2. Materials
2.1. Reagents for Cleavage of Fusion Proteins with Serine Proteases
1. Cleavage buffer: 50 mM Tris–HCl (see Note 1), 50 mM NaCl, 2 mM CaCl 2 ,(see
Note 2), pH 8.
2. Microfuge tubes.
3. Pipettes and tips for accurate liquid dispensation in the 10 μl, 100 μl and 1 ml
ranges.
4. Ice.
5. Heating block.
6. Reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–
PAGE) loading buffer, 2×: 50 mM Tris–HCl, 2% SDS, 10% glycerol. Bromophenol
blue, 0.02%, pH 6.8, or as supplied for proprietary PAGE systems.
7. Dialysis equipment (if required) for example, tubing or centrifugal concentrator.
8. HCl, 1 M.
2.2. Reagents for Cleavage of Fusion Proteins with Cysteine Proteases
1. Cleavage buffer: 50 mM Tris–HCl (see Note 1), 150 mM NaCl, 1 mM ethylenediamine
tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT) (see Note 3), pH
7.5.
2. Microfuge tubes.
3. Pipettes and tips for accurate liquid dispensation in the 10 μl, 100 μl and 1 ml
ranges.
4. Ice.
5. Reducing SDS–PAGE loading buffer, 2×: 50 mM Tris–HCl, 2% SDS, 10% glycerol.
Bromophenol blue, 0.02%, pH 6.8, or as supplied for proprietary PAGE systems.
6. Dialysis equipment (if required) for example, tubing or centrifugal concentrator.
7. HCl, 1 M.
3. Method
3.1. Selecting the Appropriate Protease
1. Based on the background information and the data in Table 1, select a protease
appropriate for the fusion protein of interest.
2. Examine the target protein amino acid sequence for complete or partial occurrences
of the recognition sequence for the intended protease. Where that sequence, or the
two or three residues around the cleavage site, exists in the target protein product,
avoid the use of that protease.
3. Insert the cleavage sequence between the fusion partner and the protein product.
4. Sequence the construct to ensure the correct insertion of the protease recognition
sequence.