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.
Original language | English (US) |
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Article number | e53845 |
Journal | Journal of Visualized Experiments |
Volume | 2016 |
Issue number | 110 |
DOIs | |
State | Published - Apr 2016 |
Keywords
- Cloning
- Crystallization
- Issue 110
- Molecular biology
- Norovirus
- P domain
- Protein expression
- Protein purification
ASJC Scopus subject areas
- General Neuroscience
- General Chemical Engineering
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology