title: Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.
abstract: The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products.
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: de la Cruz
authors.affiliation: School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
authors.forename: Marcos
authors.initials: M
authors.lastname: Alcocer
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Madrid
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Aina
authors.initials: A
authors.lastname: García
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Martín
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: González
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. Electronic address: tgarcia@vet.ucm.es.
authors.forename: Teresa
authors.initials: T
authors.lastname: García
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000276:immunology*; D055503:Protein Multimerization; D000068181:Prunus dulcis / Q000737:chemistry*; D011995:Recombination, Genetic / Q000235:genetics*; D012015:Reference Standards; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
chemical_list: D010455:Peptides; D010940:Plant Proteins; D057127:Single-Chain Antibodies
keywords: Avidin; In vivo biotinylation; Multimeric scFv; Pichia pastoris; Prunus dulcis