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.