Functional expression, purification, characterization, and membrane reconstitution of non-structural protein 2 from hepatitis C virus

Protein Expression and Purification
Marie-Laure FogeronAnja Böckmann

Abstract

Non-structural protein 2 (NS2) of the hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease and that plays a central organizing role in assembly of infectious progeny virions. While the crystal structure of the protease domain has been solved, the NS2 full-length form remains biochemically and structurally uncharacterized because recombinant NS2 could not be prepared in sufficient quantities from cell-based systems. We show here that functional NS2 in the context of the NS2-NS3pro precursor protein, ensuring NS2-NS3 cleavage, can be efficiently expressed by using a wheat germ cell-free expression system. In this same system, we subsequently successfully produce and purify milligram amounts of a detergent-solubilized form of full-length NS2 exhibiting the expected secondary structure content. Furthermore, immuno-electron microscopy analyses of reconstituted proteoliposomes demonstrate NS2 association with model membranes.

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Citations

Jul 19, 2020·Protein Expression and Purification·Johann HabersetzerSonia Fieulaine
Sep 30, 2020·Viruses·Lauriane LecoqAnja Böckmann
Apr 13, 2021·Frontiers in Molecular Biosciences·Marie-Laure FogeronAnja Böckmann
May 28, 2021·Frontiers in Molecular Biosciences·Nadide AltincekicAndreas Schlundt

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