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PLoS One. 2010 Nov 12;5(11):e13961. doi: 10.1371/journal.pone.0013961.

A versatile molecular tagging method for targeting proteins to avian reovirus muNS inclusions. Use in protein immobilization and purification.

Author information

1
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Center for Research in Biological Chemistry and Molecular Materials, University of Santiago de Compostela, Santiago de Compostela, Spain.

Abstract

BACKGROUND:

Avian reoviruses replicate in viral factories, which are dense cytoplasmic compartments established 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 self-interacting domains.

METHODOLOGY/PRINCIPAL FINDINGS:

In this study we have devised a strategy to identify muNS regions containing self-interacting 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.

PMID:
21103063
PMCID:
PMC2980485
DOI:
10.1371/journal.pone.0013961
[Indexed for MEDLINE]
Free PMC Article

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