View - ResearchGate

Site-Specific Cleavage of Fusion Proteins 215
based around a charged amino acid, as is the case with many of the other
proteases, Genenase I offers a quite different cleavage mechanism. It is tolerant
of somewhat harsher conditions than its mammalian counterparts.
Owing to the requirement for substrate-assisted catalysis, the overall activity
of this enzyme is considerably lower than other, fully self-functional proteases.
This often translates to a requirement for higher enzyme : substrate ratios. As a
licensed product, Genenase I is only available from one manufacturer and may
impose a cost limitation to future scale-up of a cleavage system.
1.6. Viral Cysteine Proteases
To obtain novel site-specific proteases, attention has turned to the enzymes
of RNA viruses. Upon infection, the genomes of these viruses are translated
as one large polyprotein (13). The proteases act to specifically cleave the
polyprotein into its individual structural and functional components. A major
feature that distinguishes this group of proteases is that they employ a cysteine
residue at the core of their catalytic mechanism, as opposed to the serine of
the mammalian and bacterial proteases. The overall fold of these viral enzymes
is very similar to that of the serine proteases; in some cases, the active site
cysteine can be substituted with serine to achieve an active enzyme, albeit with
significantly diminished activity (14).
Many viral proteases are highly specific for very long recognition sequences,
but the two that have made the greatest impact in fusion protein cleavage are
the proteases of Tobacco Etch Virus (TEV) and Human Rhinovirus (HRV).
The recognition sequence for these enzymes spans at least seven and eight
residues, respectively, with little divergence from the wild-type sequence of
the natural polyprotein junctions possible. The minimum cleavage site for TEV
protease is of the form E-X-X-Y-X-Q↓(G/S), with a consensus sequence of
E-N-L-Y-F-Q↓(G/S) (15,16). The site for HRV follows a similar general theme,
with a consensus sequence of L-E-V-L-F-Q↓G-P (17). As can be seen from
these sequences, the viral proteases cleave within their recognition sequences
and will hence leave a non-natural monopeptide or dipeptide extension on the
N terminus of the target protein. TEV protease is somewhat more flexible in its
P1´ requirements, with peptide studies suggesting that it may tolerate Glycine,
Serine, Alanine or Methionine at P1´ (18). Although for initial proof of concept
cleavage trials, it would be advisable to maintain the wild-type Glycine or
Serine.
High purity recombinant preparations of TEV and HRV proteases are
available for fusion protein cleavage. Many manufacturers’ implementations of
these enzymes also bear an affinity tag to facilitate later removal of the protease
from the protein preparation.