Expression, purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro

• Published in: Protein expression and purification 2011, Vol.75 (1), p.40-45
• Main Authors: Fuellgrabe, Marc W., Boonrod, Kajohn, Jamous, Rana, Moser, Mirko, Shiboleth, Yoel, Krczal, Gabi, Wassenegger, Michael
• Format: Article
• Language: English
• Subjects: Cucurbita - virology; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - isolation & purification; Cysteine Endopeptidases - metabolism; Escherichia coli; Escherichia coli - genetics; Gene Expression; HC-Pro; Histidine - genetics; Histidine - isolation & purification; Histidine - metabolism; Maltose-Binding Proteins - genetics; Maltose-Binding Proteins - isolation & purification; Maltose-Binding Proteins - metabolism; Mosaic Viruses - genetics; Mosaic Viruses - isolation & purification; Mosaic Viruses - metabolism; Oligopeptides - genetics; Oligopeptides - isolation & purification; Oligopeptides - metabolism; Potyviruses; Protein Binding; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; RNA, Small Interfering - metabolism; sRNA binding; Viral Proteins - genetics; Viral Proteins - isolation & purification; Viral Proteins - metabolism; Zucchini yellow mosaic virus; ZYMV
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.07.008

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.