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  • Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

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    Author(s)
    Leuthold, MM
    Koromyslova, AD
    Singh, BK
    Hansman, GS
    Griffith University Author(s)
    Hansman, Grant S.
    Year published
    2016
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    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 ...
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    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.
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    Journal Title
    Journal of Visualized Experiments (JoVE)
    Volume
    2016
    Issue
    110
    DOI
    https://doi.org/10.3791/53845
    Copyright Statement
    © 2016 The Authors. The attached file is reproduced here in accordance with the copyright policy of the publisher. Please refer to the journal's website for access to the definitive, published version.
    Subject
    Biochemistry and cell biology
    Clinical sciences
    Oncology and carcinogenesis
    Psychology
    Cognitive and computational psychology
    Science & Technology
    Multidisciplinary Sciences
    Molecular Biology
    Publication URI
    http://hdl.handle.net/10072/406814
    Collection
    • Journal articles

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