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 coil 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 (C) 2010 Elsevier Inc All rights reserved