Expression, purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro
HC-Pro is a helper component-proteinase which acts as a multifunctional protein in the potyviral life cycle. Apart from its proteolytic activity, HC-Pro has the capacity to bind duplex small RNAs (sRNAs). To investigate HC-Pro-mediated sRNA binding in vitro, high amounts of purified protein are requ...
Gespeichert in:
| Hauptverfasser: | , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
2011
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| In: |
Protein expression and purification
Year: 2011, Jahrgang: 75, Heft: 1, Pages: 40-45 |
| ISSN: | 1096-0279 |
| DOI: | 10.1016/j.pep.2010.07.008 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1016/j.pep.2010.07.008 Verlag, lizenzpflichtig, Volltext: https://www.sciencedirect.com/science/article/pii/S1046592810002135 
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| Verfasserangaben: | Marc W. Fuellgrabe, Kajohn Boonrod, Rana Jamous, Mirko Moser, Yoel Shiboleth, Gabi Krczal, Michael Wassenegger |
MARC
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| 245 | 1 | 0 | |a Expression, purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro |c Marc W. Fuellgrabe, Kajohn Boonrod, Rana Jamous, Mirko Moser, Yoel Shiboleth, Gabi Krczal, Michael Wassenegger |
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| 520 | |a HC-Pro is a helper component-proteinase which acts as a multifunctional protein in the potyviral life cycle. Apart from its proteolytic activity, HC-Pro has the capacity to bind duplex small RNAs (sRNAs). To investigate HC-Pro-mediated sRNA binding in vitro, high amounts of purified protein are required. For this purpose, the Zucchini yellow mosaic virus (ZYMV) HC-Pro was expressed as a fusion with hexa-histidine (6xHis) or maltose-binding protein (MBP) in Escherichia coli. The expressed fusion proteins were purified by affinity chromatography. 6xHis:HC-Pro and MBP:HC-Pro were partially soluble. Electrophoretic mobility-shift assays demonstrated that only MBP:HC-Pro exhibits the sRNA binding activity. The recombinant HC-Pro bound 21bp siRNAs as well as 19bp and 24bp siRNAs. A point mutation in the highly conserved FRNK box produced the HC-ProFINK protein, previously shown to be associated with reduced viral symptoms and weak sRNA binding. In this study, sRNA binding of the MBP:HA-HC-ProFINK was not detectable. The high yield of purified HC-Pro offers the possibility to study the biochemistry of the protein in detail. | ||
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