title: Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris.
abstract: The Nicotiana tabacum lectin, also called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exposure to jasmonates. Purification of the lectin from raw material is a time-consuming process, demanding large amounts of induced plant material. In addition, the lectin yield is low and purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In a way to improve and facilitate the purification of the tobacco lectin, we cloned the Nictaba gene in a vector optimized for protein expression in the methylotrophic yeast Pichia pastoris. In this report, we present data of the expression profile of recombinant Nictaba in the P. pastoris culture medium and in P. pastoris cells together with the purification strategy using ion exchange chromatography and affinity chromatography on a column with immobilized ovomucoid. Pichia transformants were estimated to express approximately 6mg of recombinant lectin per liter medium after a 72h culture. SDS-PAGE and Western blot analysis revealed that the recombinant lectin expressed in Pichia exists in two molecular forms. Edman degradation and mass spectrometry analysis confirmed the presence of at least two forms of recombinant lectin with molecular weights of 19,060 and 20,100Da, corresponding to lectin polypeptides similar to the fully processed Nictaba which is N-terminally blocked, and Nictaba extended at the N-terminus with the amino acids residues EAEAYVEFT due to incomplete processing of the alpha-factor mating sequence. Further characterisation of the recombinant lectin revealed agglutination and carbohydrate-binding properties similar to the native tobacco lectin.
authors.affiliation: Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
authors.forename: Nausicaä
authors.initials: N
authors.lastname: Lannoo
authors.forename: Wouter
authors.initials: W
authors.lastname: Vervecken
authors.forename: Paul
authors.initials: P
authors.lastname: Proost
authors.forename: Pierre
authors.initials: P
authors.lastname: Rougé
authors.forename: Els J M
authors.initials: EJ
authors.lastname: Van Damme
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000235:genetics; D020869:Gene Expression Profiling; D017343:Genes, Plant; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D037121:Plant Lectins / Q000235:genetics* / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014026:Nicotiana / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D018744:DNA, Plant; D037121:Plant Lectins; D011994:Recombinant Proteins