title: Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography.
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.
authors.affiliation: Schaller Research Group, University of Heidelberg and the German Cancer Research Center; Department of Infectious Diseases, Virology, University of Heidelberg.
authors.forename: Mila M
authors.initials: MM
authors.lastname: Leuthold
authors.affiliation: Schaller Research Group, University of Heidelberg and the German Cancer Research Center; Department of Infectious Diseases, Virology, University of Heidelberg.
authors.forename: Anna D
authors.initials: AD
authors.lastname: Koromyslova
authors.affiliation: Schaller Research Group, University of Heidelberg and the German Cancer Research Center; Department of Infectious Diseases, Virology, University of Heidelberg.
authors.forename: Bishal K
authors.initials: BK
authors.lastname: Singh
authors.affiliation: Schaller Research Group, University of Heidelberg and the German Cancer Research Center; Department of Infectious Diseases, Virology, University of Heidelberg; g.hansman@dkfz-heidelberg.de.
authors.forename: Grant S
authors.initials: GS
authors.lastname: Hansman
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray / Q000379:methods*; D004926:Escherichia coli; D006801:Humans; D029322:Norovirus / Q000737:chemistry*; D054730:Protein Interaction Domains and Motifs
chemical_list: D036022:Capsid Proteins