Production of human norovirus protruding domains in E. coli for X-ray crystallography

• Verfasst von: Leuthold, Mila [VerfasserIn]
• Verfasst von: Koromyslova, Anna D. [VerfasserIn]
• Verfasst von: Singh, Bishal [VerfasserIn]
• Verfasst von: Hansman, Grant S. [VerfasserIn]
• Titel: Production of human norovirus protruding domains in E. coli for X-ray crystallography
• Verf.angabe: Mila M. Leuthold, Anna D. Koromyslova, Bishal K. Singh, Grant S. Hansman
• E-Jahr: 2016
• Jahr: 4/19/2016
• Umfang: 1 Online-Ressource (1 Videodatei, 10:46 min)
• Umfang: 9 S.
• Illustrationen: farbig
• Fussnoten: Gesehen am 25.06.2020 ; Enthält auch Textversion ; Wissenschaftlicher Film. Deutschland. 2016
• Titel Quelle: Enthalten in: JoVE. Video journal
• Ort Quelle: [S.l.] : [s.n.], 2006
• Jahr Quelle: 2016
• Band/Heft Quelle: (2016,110) Artikel-Nummer e53845, 9 Seiten
• ISSN Quelle: 1940-087X
• DOI: doi:10.3791/53845
• Datenträger: Online-Ressource
• Dokumenttyp: Film
• Sprache: eng
• K10plus-PPN: 1702127338

Abstract

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions. The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.