title: Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish.
abstract: Hydrophilic chlorophyll (Chl)-binding proteins have been isolated from various Brassicaceae plants and are categorized into Class II water-soluble Chl-binding proteins (WSCPs). Although the molecular properties of class II WSCPs including Brassica-type (e.g., cauliflower WSCP, Brussels sprouts WSCP and BnD22, a drought- and salinity-stress-induced 22 kDa protein of rapeseed), a Lepidium-type, and an Arabidopsis-type WSCPs have been well characterized, those of Raphanus-type WSCPs are poorly understood. To gain insight into the molecular diversity of Class II WSCPs, we cloned a novel cDNA encoding a Raphanus sativus var. raphanistroides (Japanese wild radish called 'Hamadaikon') WSCP (RshWSCP). Sequence analysis revealed that the open reading frame of the RshWSCP gene consisted of 666 bp encoding 222 aa residues, including 23 residues of a deduced signal peptide. Functional recombinant RshWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (RshWSCP-His). Although the RshWSCP-His was expressed as a soluble protein in E. coli, the apo-protein was highly unstable and tended to aggregate during a series of purification steps. When the soluble fraction of RshWSCP-His-expressing E. coli was mixed immediately with homogenate of spinach leaves containing thylakoid, RshWSCP-His was able to remove Chl molecules from the thylakoid and formed a stable Chl-WSCP complex with high hydrophilicity. UV-visible absorption spectra of the reconstituted RshWSCP-His revealed that RshWSCP-His is one of the Class IIA WSCP with the highest Chl a/b ratio analyzed thus far. A semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that RshWSCP was transcribed in buds and flowers but not in roots, stems and various leaves.
authors.affiliation: Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Takahashi
authors.forename: Mayuko
authors.initials: M
authors.lastname: Ono
authors.forename: Akira
authors.initials: A
authors.lastname: Uchida
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nakayama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Satoh
mesh_terms: D001483:Base Sequence; D061146:Chlorophyll Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D035264:Flowers / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D018517:Plant Roots / Q000737:chemistry; D018547:Plant Stems / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000737:chemistry; D031224:Raphanus / Q000737:chemistry* / Q000235:genetics*; D017423:Sequence Analysis, RNA; D012995:Solubility
chemical_list: D061146:Chlorophyll Binding Proteins