title: Expression, purification, and functional characterization of a stable helicase domain from a tomato mosaic virus replication protein.
abstract: Tomato mosaic virus (genus, Tobamovirus) is a member of the alphavirus-like superfamily of positive-strand RNA viruses, which include many plant and animal viruses of agronomical and clinical importance. The RNA of alphavirus-like superfamily members encodes replication-associated proteins that contain a putative superfamily 1 helicase domain. To date, a viral three-dimensional superfamily 1 helicase structure has not been solved. For the study reported herein, we expressed tomato mosaic virus replication proteins that contain the putative helicase domain and additional upstream N-terminal residues in Escherichia coli. We found that an additional 155 residues upstream of the N-terminus of the helicase domain were necessary for stability. We developed an efficient procedure for the expression and purification of this fragment and have examined factors that affect its stability. Finally, we also showed that the stable fragment has nucleoside 5'-triphosphatase activity.
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Xiang
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ishibashi
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Nishikiori
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Mauren C
authors.initials: MC
authors.lastname: Jaudal
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ishikawa
authors.affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. Electronic address: ekatoh@nias.affrc.go.jp.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Katoh
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017898:Tobamovirus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D020365:RNA Helicases