title: Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris.
abstract: The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.
authors.affiliation: Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Belgium.
authors.forename: Bassam
authors.initials: B
authors.lastname: Al Atalah
authors.forename: Elke
authors.initials: E
authors.lastname: Fouquaert
authors.forename: Dieter
authors.initials: D
authors.lastname: Vanderschaeghe
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Jan
authors.initials: J
authors.lastname: Balzarini
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
authors.forename: Pierre
authors.initials: P
authors.lastname: Rougé
authors.forename: Yi
authors.initials: Y
authors.lastname: Lasanajak
authors.forename: Nico
authors.initials: N
authors.lastname: Callewaert
authors.forename: Els J M
authors.initials: EJ
authors.lastname: Van Damme
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D037241:Mannose-Binding Lectins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D010843:Pichia / Q000235:genetics*; D037121:Plant Lectins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012136:Respiratory Syncytial Viruses / Q000187:drug effects; D017386:Sequence Homology, Amino Acid; D013312:Stress, Physiological
chemical_list: D019380:Anti-HIV Agents; D000998:Antiviral Agents; D018744:DNA, Plant; D037241:Mannose-Binding Lectins; D037121:Plant Lectins; D011993:Recombinant Fusion Proteins; C043696:jacalin; C500656:morniga M; C417580:orysata protein, Oryza sativa