title: Expression, purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro.
abstract: 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 21 bp siRNAs as well as 19 bp and 24 bp siRNAs. A point mutation in the highly conserved FRNK box produced the HC-Pro(FINK) protein, previously shown to be associated with reduced viral symptoms and weak sRNA binding. In this study, sRNA binding of the MBP:HA-HC-Pro(FINK) was not detectable. The high yield of purified HC-Pro offers the possibility to study the biochemistry of the protein in detail.
authors.affiliation: RLP-AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, 67435 Neustadt, Germany.
authors.forename: Marc W
authors.initials: MW
authors.lastname: Fuellgrabe
authors.forename: Kajohn
authors.initials: K
authors.lastname: Boonrod
authors.forename: Rana
authors.initials: R
authors.lastname: Jamous
authors.forename: Mirko
authors.initials: M
authors.lastname: Moser
authors.forename: Yoel
authors.initials: Y
authors.lastname: Shiboleth
authors.forename: Gabi
authors.initials: G
authors.lastname: Krczal
authors.forename: Michael
authors.initials: M
authors.lastname: Wassenegger
mesh_terms: D028464:Cucurbita / Q000821:virology; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009029:Mosaic Viruses / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D006639:Histidine; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus