title: Expression and purification of a biologically active Phytophthora parasitica cellulose binding elicitor lectin in Pichia pastoris.
abstract: The Phytophthora parasitica cellulose-binding elicitor lectin, (CBEL), is a cell wall-localized protein playing a key role in cell wall organization and adhesion of the mycelium to cellulosic substrates. CBEL is a potent elicitor of plant immune responses and this activity is linked to its ability to bind plant cell wall components. In order to scale up the production of active CBEL, we reported here the cloning and expression of a His-tagged version of CBEL in the yeast Pichia pastoris. Selection of a high-producing P. pastoris clone and optimization of the purification procedure allowed a yield of about 2mg of pure protein per liter of culture filtrate. The identity of the recombinant protein was confirmed by western-blot analysis, N-terminal protein sequencing, and by peptide mass fingerprinting. The cellulose-binding affinity and the lectin activity of the recombinant protein were identical to the native CBEL. Its elicitor activity, tested on Arabidopsis thaliana leaves, was similar to the native CBEL protein as it displays a similar biological activity on plant immune responses inducing defense gene expression and localized necroses of the infiltrated leaf tissues. The present work suggests that P. pastoris can be a suitable host for the production of compounds active on plants or for the development of new agricultural products able to stimulate plant immunity.
authors.affiliation: Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, UPS, 24 Chemin de Borde Rouge, BP42617, Auzeville F-31326, Castanet-Tolosan, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Larroque
authors.forename: Diana
authors.initials: D
authors.lastname: Ramirez
authors.forename: Claude
authors.initials: C
authors.lastname: Lafitte
authors.forename: Gisèle
authors.initials: G
authors.lastname: Borderies
authors.forename: Bernard
authors.initials: B
authors.lastname: Dumas
authors.forename: Elodie
authors.initials: E
authors.lastname: Gaulin
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D037102:Lectins / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010838:Phytophthora / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology; D018515:Plant Leaves / Q000276:immunology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D003470:Culture Media; D037102:Lectins; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C110312:elicitor protein, Phytophthora parasitica; D006639:Histidine; D002482:Cellulose