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

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 f...

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Bibliographic Details
Main Authors: Leuthold, Mila (Author) , Koromyslova, Anna D. (Author) , Singh, Bishal (Author) , Hansman, Grant S. (Author)
Format: Article (Journal) Video
Language:English
Published: 4/19/2016
In: JoVE. Video journal
Year: 2016, Issue: 110, Pages: 1-9
ISSN:1940-087X
DOI:10.3791/53845
Subjects:
Film
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.3791/53845
Verlag, lizenzpflichtig, Volltext: https://www.jove.com/video/53845/production-human-norovirus-protruding-domains-e-coli-for-x-ray
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Author Notes:Mila M. Leuthold, Anna D. Koromyslova, Bishal K. Singh, Grant S. Hansman
Description
Summary: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.
Item Description:Gesehen am 25.06.2020
Enthält auch Textversion
Wissenschaftlicher Film. Deutschland. 2016
Physical Description:Online Resource
ISSN:1940-087X
DOI:10.3791/53845

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