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226 Charlton
22. The full-length fusion protein and the separated affinity tag will bind to the
affinity column under the same conditions employed to generate the fusion protein
initially. The correctly cleaved protein, now lacking an affinity tag, will not be
bound by the column and will thus flow-through. It should be noted that internal
cleavage fragments of the product (if generated) will not be separated by this
technique. If an internally cut protein is held together by disulphide bonds (see
Note 12), it may be successfully separated from intact protein by ion-exchange
chromatography due to the extra surface charges provided by the hydrolysis
sites. Where the internal cleavage fragments are not held together, size-exclusion
chromatography may provide separation.
23. Zinc ions are quite potent inhibitors of cysteine protease activity, with concentrations
as low as 5 mM resulting in significant loss of activity. This inactivation
is thought to occur due to the formation of a complex between the zinc ion and
three amino acids in the active site pocket, including the catalytic cysteine (21).
24. Although not the optimal temperature for these enzymes, it has been shown, at
least in the case of TEV protease, that incubation at 4°C results in only a 3-fold
reduction in overall activity compared to room temperature (20°C) (30). The
benefit to product stability at low temperature is, in most cases, well worth a
slightly longer incubation time.
25. Internal cleavage by viral cysteine proteases is highly unlikely, with no reported
cleavage at sites other than the minimum penta- or hexa-peptide recognition
sequences in fusion proteins.
26. Degradation may be due to the action of bacterial host proteases that have copurified
with the fusion protein.
27. Viral proteases are far more salt tolerant than the serine proteases with activity
reported in 800 mM NaCl (18).
References
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2. Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. and Nygren, P. A. (1997). Affinity
fusion strategies for detection, purification, and immobilization of recombinant
proteins. Protein Expr. Purif. 11, 1–16.
3. Schechter, I. and Berger, A. (1967). On the size of the active site in proteases. I.
Papain. Biochem. Biophys. Res. Commun. 27, 157–162.
4. Maroux, S., Baratti, J. and Desnuelle, P. (1971). Purification and specificity of
porcine enterokinase. J. Biol. Chem. 246, 5031–5039.
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