cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris.
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.
Almeida
M S
MS
Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, RJ, Brazil.
Cabral
K S
KS
de Medeiros
L N
LN
Valente
A P
AP
Almeida
F C
FC
Kurtenbach
E
E
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
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