Isolation of scFvs that Inhibit the NS3 Protease of Hepatitis C Virus by a Combination of Phage Display and a Bacterial Genetic
The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has been implicated in interactions with various cell constituents, resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for antiviral drugs. We established a genetic screen that is based on coexpression of NS3, a β-galactosidase reporter that is cleavable by NS3, and potential inhibitors within the same bacterial cell. A single-chain antibody (scFv) library was prepared from spleens of NS3-immunized mice and the screen was used to isolate a panel of protease-inhibiting scFvs. Candidate scFvs were validated for inhibitory activity using an o -nitrophenyl-β-galactoside (ONPG) hydrolysis assay. The methods can be used more generally to isolate protease-inhibiting cytoplasmic intrabodies able to inhibit proteases or other activities that can be linked with the phenotype of Escherichia coli .
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