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recognition sequence, rather than within it. This therefore provides the opportunity
to generate the exact sequence for the target protein, as no contribution
to catalysis needs to be made by any element of the target protein itself.
A very limited number of all proteases display suitable site specificity for a
sufficiently long amino acid sequence to be useful for fusion protein cleavage.
These proteases are frequently isolated as proprotein activation enzymes, where
evolutionary pressure has led toward site specificity. This is the case with many
of the proteases covered in this chapter, which represent those that are both
readily commercially available and have a long history of application in fusion
protein cleavage.
1.1. Fusion Proteins
The fusion protein strategy is a popular approach to the expression of recombinant
proteins in bacteria. The fusion of the protein of interest to another
unrelated protein, or fusion partner can improve yields of the target protein. The
fusion partner can provide protection against proteolysis, enable in vivo folding
of the target protein or facilitate recovery by acting as affinity tags (1,2).
The protease substrate numbering convention of Schechter and Berger (3)
will be used for this chapter, where the amino acids of the substrate (the fusion
protein) N terminal to the site of cleavage are designated P and those C terminal
are P´. The residues are numbered with increasing distance from the scissile
bond (see Fig. 1).
The fusion partner may be incorporated at the N- or C-terminal end of the
target protein, but for the purposes of this chapter, N-terminal fusions will
be specifically covered. As all the specific proteases detailed cleave on the
carbonyl side of the P1 residue, less or no non-native sequence elements are
retained from these fusions. The methods are valid for C-terminal fusions, but
the recognition sequences will remain attached as a C-terminus extension of
the protein product. Figure 1 depicts an N-terminal fusion protein.
Fig. 1. A schematic representation of an N-terminal fusion protein.