Zusammenfassung
Affinity tags have become indispensable tools for protein expression
and purification. Yet, because they have the potential to interfere
with structural and functional studies, it is usually desirable
to remove them from the target protein. The stringent sequence specificity
of the tobacco etch virus (TEV) protease has made it a useful reagent
for this purpose. However, a potential limitation of TEV protease
is that it is believed to require a Gly or Ser residue in the P1'
position of its substrates to process them with reasonable efficiency.
Consequently, after an N-terminal affinity tag is removed by TEV
protease, the target protein will usually retain a non-native Ser
or Gly residue on its N-terminus, and in some cases this may affect
its biological activity. To investigate the stringency of the requirement
for Gly or Ser in the P1' position of a TEV protease recognition
site, we constructed 20 variants of a fusion protein substrate with
an otherwise optimal recognition site, each containing a different
amino acid in the P1' position. The efficiency with which these
fusion proteins were processed by TEV protease was compared both
in vivo and in vitro. Additionally, the kinetic parameters K(M)
and k(cat) were determined for a representative set of peptide substrates
with amino acid substitutions in the P1' position. The results indicate
that many side-chains can be accommodated in the P1' position of
a TEV protease recognition site with little impact on the efficiency
of processing.
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