Isolation of scFvs that inhibit the NS3 protease of hepatitis C virus by a combination of phage display and a bacterial genetic screen.
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 beta-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-beta-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.
Nahary
Limor
L
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, 69978, Israel.
Trahtenherts
Alla
A
Benhar
Itai
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology / Q000494:pharmacology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005822:Genetic Vectors; D016174:Hepacivirus / Q000201:enzymology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D012689:Sequence Homology, Nucleic Acid; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D017361:Viral Nonstructural Proteins; D001616:beta-Galactosidase; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases