Protein Engineering, Vol 11, 1257-1265, Copyright © 1998 by Oxford University Press
N Dimasi, A Pasquo, F Martin, S Di Marco, C Steinkuhler, R Cortese and M Sollazzo
The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed
into functional polypeptides by both host- and virus-encoded proteases. The
HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent
complex, which participates in processing the viral polyprotein. This
proteolytic activity is believed to be essential for virus proliferation
and thus the NS3 protease is a prime target for developing anti-HCV
pharmacological agents. Recent X-ray crystallography structural studies
have revealed the nature of this non- covalent complex between NS3 protease
and the 'active' central segment of NS4A, providing the opportunity to
design a single-chain polypeptide. To this end, the DNA sequence encoding
for the NS4A peptide (residues 21-34) was genetically fused via a short
linker, capable of making a beta-turn, to the N-terminus of the NS3
protease domain. This engineered single-chain NS3-protease (scNS3) is fully
active with kinetic parameters virtually identical with those of the NS3/
NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on
filamentous phage and affinity selected using an immobilized specific
inhibitor. The scNS3 expressed as a soluble protein and in a phage-display
format facilitates enzyme engineering for further structural studies and in
vitro selection of potential drug- resistant mutants. These are important
steps towards developing effective anti-protease compounds.
ARTICLES
Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein
Istituto di Ricerche di Biologia Molecolare, Pomezia (Rome), Italy.
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