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214 Charlton
when selecting Factor Xa for a fusion protein system, depending on the intended
final use of the target protein product.
1.4. Thrombin
Thrombin (EC 3.4.21.5) is another enzyme of the mammalian blood clotting
cascade, acting downstream of Factor Xa its function in vivo is the cleavage of
fibrinogen to generate fibrin (8). Unlike the other specific proteases described
in this chapter, thrombin does not have a long defined specificity sequence,
with the only absolute requirement for cleavage being that it occurs after an
Arginine, especially where the Arginine residue is preceded by a Glycine or a
Proline at P2 and followed by a Glycine at P1´ (9). Although lacking a long
recognition sequence, thrombin cleavage can be further targeted by inclusion of
hydrophobic residues in the P4 and P3 positions (9). Thrombin cleavage is also
improved with non-acidic P1´ and P2´ residues, but these will be determined
by the target protein’s sequence and not usually available for substitution.
Thrombin distinctly prefers cleavage within a P-R↓G sequence, so much so
that it should be considered to cleave within this recognition sequence, and
as such a protein released from a fusion by this protease will have a residual
N-terminal Glycine. Thrombin is therefore unlikely to produce the target protein
with fully authentic sequence, except in cases where the first residue of the
protein is Glycine. There are examples of thrombin cleavage prior to residues
other than Glycine, but these are uncommon (10).
Thrombin possesses high intrinsic activity, so can function at relatively low
enzyme concentrations and is tolerant of a wider range of buffer conditions
than other mammalian proteases. Like Factor Xa, thrombin is not commercially
available as a recombinant product, so consideration of the purpose for the
target protein must be made before designing a fusion protein regime around
this protease.
1.5. Genenase I
Genenase I is unique amongst the selected proteases, as it represents the only
example of a bacterial enzyme and of a protease with engineered specificity.
The parent enzyme for this rationally designed protease is subtilisin BPN´ from
the bacterium Bacillus subtilis (11). Genenase I was developed by mutation of a
necessary active site Histidine residue to Alanine, resulting in a non-functional
enzyme. The functionality of the protease can be restored if the side chain of
the Histidine residue is supplied by the substrate at the P2 or P1´ position; this
mechanism is known as substrate-assisted catalysis (11,12).
Cleaving C terminal to its ideal recognition sequence, Genenase I is capable
of producing the correct N terminus for the product. As this sequence is not