title: Development of an influenza virus protein array using Sortagging technology.
abstract: Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.
authors.affiliation: Whitehead Institute for Biomedical Research , 9 Cambridge Center, Cambridge, Massachusetts 02142, United States.
authors.forename: Antonia
authors.initials: A
authors.lastname: Sinisi
authors.forename: Maximilian Wei-Lin
authors.initials: MW
authors.lastname: Popp
authors.forename: John M
authors.initials: JM
authors.lastname: Antos
authors.forename: Werner
authors.initials: W
authors.lastname: Pansegrau
authors.forename: Silvana
authors.initials: S
authors.lastname: Savino
authors.forename: Mikkel
authors.initials: M
authors.lastname: Nissum
authors.forename: Rino
authors.initials: R
authors.lastname: Rappuoli
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: Ludovico
authors.initials: L
authors.lastname: Buti
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D006389:Hemagglutinins, Viral / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007251:Influenza, Human / Q000821:virology; D040081:Protein Array Analysis / Q000295:instrumentation*; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006389:Hemagglutinins, Viral; D055767:Immobilized Proteins; D014764:Viral Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases