Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease

Cabral, Katia M. S.; Almeida, Marcius S.; Valente, Ana Paula; Almeida, Fábio C. L.; Kurtenbach, Eleonora

doi:10.1016/s1046-5928(03)00136-0

Cited by 91 publications

(57 citation statements)

References 24 publications

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“…Expression of NaD1 and NaD1 Mutants in Pichia pastorisNaD1 and NaD1 mutants were recombinantly expressed in the methylotrophic yeast P. pastoris essentially as described by Cabral et al (50). Briefly, DNA encoding the mature region of NaD1 was cloned into the pPIC9 expression vector (Invitrogen) directly in-frame with the ␣-mating factor secretion signal using the restriction enzymes XhoI and NotI.…”

Section: Methodsmentioning

confidence: 99%

Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity

Lay

Mills

Poon

et al. 2012

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity

Lay

Mills

Poon

et al. 2012

Journal of Biological Chemistry

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“…To meet this goal, we selected methylotrophic yeast P. pastoris as an excellent alternative to the E. coli expression system for successful production of a disulfide-rich SN-1 peptide. This yeast was chosen in part because P. pastoris cells under the control of a methanol-induced AOX1 promoter can produce large amounts of functionally active cysteine-rich AMPs [30,31] that are secreted directly into the culture media [32]. We then constructed an efficient P. pastoris expression system for production of the recombinant SN-1.…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

Kuddus

Rumi

Tsutsumi

et al. 2016

Protein Expression and Purification

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Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum).Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris.A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and 1 H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active 3 SN-1 in P. pastoris.4

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“…They have been identified in diverse taxa with many defense roles, including antifungal (Gao et al, 2000;Park et al, 2002;Cabral et al, 2003;Lay et al, 2003a), antibacterial (Osborn et al, 1995;Segura et al, 1998;Koike et al, 2002), anti-insect (Chen et al, 2002;Lay et al, 2003b), and protease inhibitory (Osborn et al, 1995;Wijaya et al, 2000) activities. In many cases, small differences in amino acid sequence can predict the specificity of the defense role (GarciaOlmedo et al, 1998).…”

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confidence: 99%

Genome Organization of More Than 300 Defensin-Like Genes in Arabidopsis

et al. 2005

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Defensins represent an ancient and diverse set of small, cysteine-rich, antimicrobial peptides in mammals, insects, and plants. According to published accounts, most species' genomes contain 15 to 50 defensins. Starting with a set of largely nodulespecific defensin-like sequences (DEFLs) from the model legume Medicago truncatula, we built motif models to search the nearcomplete Arabidopsis (Arabidopsis thaliana) genome. We identified 317 DEFLs, yet 80% were unannotated at The Arabidopsis Information Resource and had no prior evidence of expression. We demonstrate that many of these DEFL genes are clustered in the Arabidopsis genome and that individual clusters have evolved from successive rounds of gene duplication and divergent or purifying selection. Sequencing reverse transcription-PCR products from five DEFL clusters confirmed our gene predictions and verified expression. For four of the largest clusters of DEFLs, we present the first evidence of expression, most frequently in floral tissues. To determine the abundance of DEFLs in other plant families, we used our motif models to search The Institute for Genomic Research's gene indices and identified approximately 1,100 DEFLs. These expressed DEFLs were found mostly in reproductive tissues, consistent with our reverse transcription-PCR results. Sequence-based clustering of all identified DEFLs revealed separate tissue-or taxon-specific subgroups. Previously, we and others showed that more than 300 DEFL genes were expressed in M. truncatula nodules, organs not present in most plants. We have used this information to annotate the Arabidopsis genome and now provide evidence of a large DEFL superfamily present in expressed tissues of all sequenced plants.Organisms are constantly confronted with potentially pathogenic microorganisms. Yet, few encounters result in disease, due to the multilayered lines of defense each organism possesses. In vertebrates, adaptive immunity has long held center stage because of its ability to recognize almost any foreign antigen. The ancient innate immune system is equally important and provides a critical line of defense in vertebrates, invertebrates, plants, and insects (Thomma et al., 2002;Beutler, 2004;Bulet et al., 2004;Finlay and Hancock, 2004).In plants, innate immunity occurs via elaborate mechanisms (Dangl and Jones, 2001;Veronese et al., 2003). The plant cell wall serves as a barrier to microbial penetration. Antimicrobial compounds deter would-be invaders. Should penetration occur, recognition leads to the production of reactive oxygen intermediates, cell wall strengthening, activation of protein kinase pathways, and the production of signaling intermediates. Signaling events lead to localized responses such as a hypersensitive response (programmed cell death) or to the release of antimicrobial compounds. The plant is also immunized against unrelated pathogens via systemic acquired resistance (Delaney, 1997;Dong, 2001;Gozzo, 2003).Much attention has focused on the interaction of plant resistance genes (R-genes) and pathoge...

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Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease

Cited by 91 publications

References 24 publications

Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity

Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris

Genome Organization of More Than 300 Defensin-Like Genes in Arabidopsis

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