a versatile molecular tagging method for targeting proteins to avian reovirus muns inclusions. use in protein immobilization and purification通用的分子标记方法针对蛋白质禽呼肠孤病毒蒙斯夹杂物。.pdfVIP

A Versatile Molecular Tagging Method for Targeting Proteins to Avian Reovirus muNS Inclusions. Use in Protein Immobilization and Purification ˜ Alberto Brandariz-Nunez, Rebeca Menaya-Vargas, Javier Benavente, Jose Martinez-Costas* 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 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 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