Expression,purification and functional characterization of recombinant Zucchini yellow mosaic virus HC-Pro |
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| Authors: | Marc W Fuellgrabe Kajohn Boonrod Rana Jamous Mirko Moser Yoel Shiboleth Gabi Krczal Michael Wassenegger |
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| Institution: | 1. RLP-AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, 67435 Neustadt, Germany;2. Biodiversity and Environmental Research Center, Til Nablus, POB 696, Palestinian Authority;3. Research Organization, Volcani Center, POB 6, Bet Dagan 50250, Israel;4. Heidelberg Institute for Plant Science, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany;5. University of Michigan, Department of Molecular, Cellular and Developmental Biology, 830 N. University, Ave4053, Natural Science Building, Ann Arbor, MI 48109-1048, USA;1. Centre for Integrative Biology (CBI), Department of Integrated Structural Biology, Institute of Genetics and of Molecular and Cellular Biology (IGBMC), 1 rue Laurent Fries, Illkirch, France;2. Centre National de la Recherche Scientifique (CNRS) UMR 7104, Illkirch, France;3. Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Illkirch, France;4. Université de Strasbourg, Strasbourg, France;1. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;2. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;1. School of Plant Protection, Anhui Agricultural University, Hefei 230036, P.R.China;2. Biotechnology Center, Anhui Agricultural University, Hefei 230036, P.R.China;3. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310000, P.R.China;3. Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada;5. Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada;4. Centres for Research in Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada;6. Research in Mass Spectrometry, York University, Toronto, Ontario M3J 1P3, Canada |
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| 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-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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