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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...

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Main Authors: Füllgrabe, Marc W. (Author) , Boonrod, Kajohn (Author) , Jamous, Rana (Author) , Moser, Mirko (Author) , Shiboleth, Yoel (Author) , Krczal, Gabi (Author) , Wassenegger, Michael (Author)
Format: Article (Journal)
Language:English
Published: 2011
In: Protein expression and purification
Year: 2011, Volume: 75, Issue: 1, Pages: 40-45
ISSN:1096-0279
DOI:10.1016/j.pep.2010.07.008
Online Access: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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Author Notes:Marc W. Fuellgrabe, Kajohn Boonrod, Rana Jamous, Mirko Moser, Yoel Shiboleth, Gabi Krczal, Michael Wassenegger
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Summary: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.
Item Description:Gesehen am 31.05.2023
Online verfügbar 30 Juli 2010
Physical Description:Online Resource
ISSN:1096-0279
DOI:10.1016/j.pep.2010.07.008

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