title: cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris.
abstract: In the present work, we describe the cDNA cloning, expression in Pichia pastoris, purification, and characterization of the recombinant Pisum sativum defensin 1 (rPsd1), a novel Cys-rich protein presenting four disulfide bridges and high antifungal activity. Several parameters that affect the level of protein expression were assayed. The best condition yielded 13.8 mg/L (1.50 microg/10(8) cells) of active rPsd1. The recombinant rPsd1 was purified to homogeneity by cation exchange, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region. Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein has a very similar folding and a correct disulfide-bonding pattern when compared to native Psd1. Nevertheless, the rPsd1 presented a more species-specific antifungal activity. The importance of the N- and C-termini for Psd1 activity is pointed out.
authors.affiliation: Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, RJ, Brazil.
authors.forename: M S
authors.initials: MS
authors.lastname: Almeida
authors.forename: K S
authors.initials: KS
authors.lastname: Cabral
authors.forename: L N
authors.initials: LN
authors.lastname: de Medeiros
authors.forename: A P
authors.initials: AP
authors.lastname: Valente
authors.forename: F C
authors.initials: FC
authors.lastname: Almeida
authors.forename: E
authors.initials: E
authors.lastname: Kurtenbach
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism*; D023082:Defensins; D004220:Disulfides; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D000935:Antifungal Agents; D018076:DNA, Complementary; D023082:Defensins; D004220:Disulfides; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C410643:defensin 1, Pisum sativum; D003545:Cysteine