title: Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease.
abstract: Plant defensins are small cysteine-rich proteins that present high activity against fungi and bacteria and inhibition of insect proteases and alpha-amylases. Here, we present the expression in Pichia pastoris, purification and characterization of the recombinant Pisum sativum defensin 1(rPsd1); a pea defensin which presents four disulfide bridges and high antifungal activity. For this, we had to overcome the inefficiency of the STE13 protease. Our strategy was to clone the corresponding cDNA directly in-frame with a variant of the widely used secretion signal from the Saccharomyces cerevisiae alpha-mating factor, devoid of the STE13 proteolytic signal cleavage sequence. Using an optimized expression protocol, which included a buffered basal salt media formulation, it was possible to obtain about 63.0mg/L of 15N-labeled and unlabeled rPsd1. The recombinants were purified to homogeneity by gel filtration chromatography, followed by reversed-phase HPLC. Mass spectrometry of native and recombinant Psd1 revealed that the protein expressed heterologously was post-translationally processed to the same mature protein as the native one. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein had the same folding when compared to native Psd1. In addition, the rPsd1 was fully active against Aspergillus niger, if compared with native Psd1. To our knowledge, this is the first heterologous expression of a fully active plant defensin in a high-yield flask.
authors.affiliation: Departamento de Bioqui;mica Médica, CCS/ICB/UFRJ, CEP: 21941-590, Rio de Janeiro, RJ, Brazil.
authors.forename: Kátia M S
authors.initials: KM
authors.lastname: Cabral
authors.forename: Marcius S
authors.initials: MS
authors.lastname: Almeida
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Valente
authors.forename: Fábio C L
authors.initials: FC
authors.lastname: Almeida
authors.forename: Eleonora
authors.initials: E
authors.lastname: Kurtenbach
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D001234:Aspergillus niger / Q000187:drug effects; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics; D023082:Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D020128:Inhibitory Concentration 50; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D000935:Antifungal Agents; D018076:DNA, Complementary; D023082:Defensins; C048632:MF(ALPHA)1 protein, S cerevisiae; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C410643:defensin 1, Pisum sativum; D000432:Methanol