SpringerProtocols: Abstract: Isolation of scFvs that Inhibit the NS3 Protease of Hepatitis C Virus by a Combination of Phage Display and a Bacterial Genetic Screen

9. Isolation of scFvs that Inhibit the NS3 Protease of Hepatitis C Virus by a Combination of Phage Display and a Bacterial Genetic Screen

By: Limor Nahary², Alla Trahtenherts², Itai Benhar²

Abstract

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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.

Affiliation(s): (2) Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, 69978, Israel

Book Title: Antibody Phage Display: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 562 | Pub. Date: Mar-01-2009 | Page Range: 115-132 | DOI: 10.1007/978-1-60327-302-2_9

Subject: Microbiology

Key Words: Immune antibody phage display library - Single-chain antibodies - Protease inhibitors - Genetic screen - Hepatitis C virus - NS3 protease - β-Galactosidase

References

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