A Versatile Molecular Tagging Method for Targeting Proteins to Avian Reovirus muNS Inclusions. Use in Protein Immobilization and Purification
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Título: | A Versatile Molecular Tagging Method for Targeting Proteins to Avian Reovirus muNS Inclusions. Use in Protein Immobilization and Purification |
Autor/a: | Brandariz Núñez, Alberto Menaya Vargas, Rebeca Benavente Martínez, Francisco Javier Martínez Costas, José Manuel |
Centro/Departamento: | Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular |
Palabras chave: | Cytoplasmic inclusions | Green fluorescent protein | Plasmid construction | Polymerase chain reaction | Protein expression | Baculoviruses | Birds | Insects | |
Data: | 2010 |
Editor: | PLOS |
Cita bibliográfica: | Brandariz-Nuñez, A., Menaya-Vargas, R., Benavente, J., & Martinez-Costas, J. (2010). A versatile molecular tagging method for targeting proteins to avian reovirus muNS inclusions. Use in protein immobilization and purification. PLoS One, 5(11). |
Resumo: | Background: Avian reoviruses replicate in viral factories, which are dense cytoplasmic compartments estabilished by protein-protein interactions. The non-structural protein muNS forms the factory scaffold that attracts other viral components in a controlled fashion. To create such a three-dimensional network, muNS uses several different selfinteracting domains. Methodology/Principal Findings: In this study we have devised a strategy to identify muNS regions containing selfinteracting domains, based on the capacity of muNS-derived inclusions to recruit muNS fragments. The results revealed that the muNS region consisting of residues 477–542 was recruited with the best efficiency, and this raised the idea of using this fragment as a molecular tag for delivering foreign proteins to muNS inclusions. By combining such tagging system with our previously established method for purifying muNS inclusions from baculovirus-infected insect cells, we have developed a novel protein purification protocol. Conclusions/Significance: We show that our tagging and inclusion-targeting system can be a simple, versatile and efficient method for immobilizing and purifying active proteins expressed in baculovirus-infected cells. We also demonstrate that muNS inclusions can simultaneously recruit several tagged proteins, a finding which may be used to generate protein complexes and create multiepitope particulate material for immunization purposes. |
Versión do editor: | https://doi.org/10.1371/journal.pone.0013961 |
URI: | http://hdl.handle.net/10347/22844 |
DOI: | 10.1371/journal.pone.0013961 |
E-ISSN: | 1932-6203 |
Dereitos: | © 2010 Brandariz-Nuñez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited |
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