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Site-Specific Cleavage of Fusion Proteins 213
In the design of a fusion protein strategy, the selection of the protease to
affect the final cleavage may be as important as the selection of the fusion
partner itself. Where available, sequence and structural information can guide
this decision, as can the final application of the target protein. When a protease
has been selected, the recognition sequence for that protease must be inserted
between the fusion partner and the target protein as a linker peptide, as shown
in Fig. 1.
1.2. Enterokinase
Enterokinase (EC 3.4.21.9) is a mammalian gastric serine protease. The
in vivo function of this enzyme is the activation of trypsin by cleavage of
the trypsinogen zymogen to its active form. The cleavage site for this enzyme
with its natural substrate is C terminal to the recognition sequence pentapeptide
(Aspartate) 4 –Lysine (4). As Enterokinase cuts C terminal to its recognition
sequence, without requiring the interaction of residues on the other side of the
scissile bond, it is capable of generating a native N terminus for the protein
product. The high charge density of the recognition sequence will increase the
likelihood of solvent exposure at the site, maximizing protease accessibility
and also serving to improve the overall solubility of the fusion protein (5).
The unique nature of the cleavage motif should preclude its occurrence
within a protein product; however, Enterokinase largely recognizes the charge
density of its recognition sequence rather than the precise amino acid sequence.
Cleavage by Enterokinase is possible down to sequences as short as Asp-Asp-
Lys (4), and activity is permitted with substitution of the motif residues with
their charge equivalents (6). Therefore, similar apparent charge densities in the
target protein may also be susceptible to Enterokinase cleavage.
Enterokinase is available as a recombinant enzyme, in many cases, as only
the catalytic subunit of the holoenzyme. It must be noted that not all vendors
offer the recombinant protein, so care must be taken in obtaining the enzyme
if this is important.
1.3. Factor Xa
Factor Xa (EC 3.4.21.6) is an enzyme of the mammalian blood clotting
cascade. Upon its own activation, this enzyme in turn activates the next
enzyme in the cascade by cleavage of prothrombin, liberating active Thrombin.
Factor Xa is highly specific for cleavage following the tetrapeptide sequence
Isoleucine–(Glutamate/Aspartate)–Glycine–Arginine, allowing for the generation
of an authentic N terminus for the protein product (7).
Factor Xa is not currently produced recombinantly and, therefore, must be
isolated from mammalian plasma (usually bovine). This should be considered