Expression of functional <i>Raphanus sativus</i> antifungal protein in yeast

Alves, Ana L. Vilas; Samblanx, Genoveva W. De; Terras, Franky R. G.; Cammue, Bruno P. A.; Broekaert, Willem F.

doi:10.1016/0014-5793(94)00597-4

Cited by 29 publications

(18 citation statements)

References 12 publications

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“…Based on these homologies it may be possible to identify conserved residues which are important to the biological activities of the different classes of peptide. Recently, we have successfully expressed functional Rs-AFP2 in Saccharomyces cerevisiae [20] and this system is being used to express variants of the peptide which have single amino acid substitutions based on differences with yl-purothionin.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

et al. 1995

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From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and ~/-thionins from Poaceae seeds. Based on the spectrum of their antimicriobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different a-amylases.

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Section: Discussionmentioning

confidence: 99%

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

et al. 1995

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“…Heterologous Expression and Purification of Rs-AFP2 VariantsTransformation of S. cerevisiae, growth of the yeast cultures, and purification of the Rs-AFP2 variants from the culture supernatants were essentially done as described previously for native Rs-AFP2 (20). Briefly, 250 ml of culture supernatant (minimal selective SD medium: 0.8 g/liter CSM-URA from BIO 101, La Jolla, CA; 6.5 g/liter yeast nitrogen base from Difco; 20 g/liter glucose (Merck); 5 g/liter casamino acids from Difco) was passed over an anion-exchange chromatography column (Q-Sepharose Fast Flow, Pharmacia) connected on-line with a disposable reversed phase C 8 silica column (Bond Elut, 500 mg solid phase, Varian, Harbor City, CA).…”

Section: Methodsmentioning

confidence: 99%

“…In a second PCR, this elongated fragment was amplified by primer OWB61, binding to the 5Ј end of the Rs-AFP2 gene, and OWB36, an oligonucleotide identical to the 5Ј tag of OWB35 (28 cycles; 1 min at 94°C, 1 min at 55°C, 1 min at 72°C). OWB61 contains a restriction site allowing in-frame cloning into the HindIII site in the MF␣1 pro-sequence region of pVD4 (20). Amplification products of the second PCR were digested with HindIII-BamHI and introduced in the corresponding sites of pVD4.…”

Section: Methodsmentioning

confidence: 99%

“…The different Rs-AFP2 analogues were purified from the yeast culture supernatant by a combination of ion-exchange chromatography and reversed-phase chromatography. Using this approach, we have previously shown that wild-type Rs-AFP2 can be produced in a correctly processed and bioactive form in yeast (20). In total, 19 Rs-AFP2 variants were produced and purified in this way (see Table II).…”

Section: Conception and Production Of Rs-afp2 Variants-proteinsmentioning

confidence: 99%

“…For the production of the different Rs-AFP2 variants with the desired amino acid substitution, the Rs-AFP2 coding sequence was mutated site-specifically by PCR, fused in frame to the yeast mating factor ␣1 (MF␣1) promoter and prepro-sequence (20,21) and subsequently transferred to yeast via a yeast shuttle vector. The different Rs-AFP2 analogues were purified from the yeast culture supernatant by a combination of ion-exchange chromatography and reversed-phase chromatography.…”

Section: Conception and Production Of Rs-afp2 Variants-proteinsmentioning

confidence: 99%

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Mutational Analysis of a Plant Defensin from Radish (Raphanus sativus L.) Reveals Two Adjacent Sites Important for Antifungal Activity

Samblanx

Goderis

Thevissen

et al. 1997

Journal of Biological Chemistry

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162

132

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Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI ␤-turn connecting ␤-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting ␤-strand 1 and the ␣-helix and contiguous residues on the ␣-helix and ␤-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca 2؉ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca 2؉ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca 2؉ uptake.

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The three-dimensional solution structure ofAesculus hippocastanum antimicrobial protein 1 determined by1H nuclear magnetic resonance

Fant¹,

Vranken²,

Borremans³

1999

Proteins

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Aesculus hippocastanum antimicrobial protein 1 (Ah-AMP1) is a plant defensin isolated from horse chestnuts. The plant defensins have been divided in several subfamilies according to their amino acid sequence homology. Ah-AMP1, belonging to subfamily A2, inhibits growth of a broad range of fungi. So far, a three-dimensional structure has been determined only for members of subfamilies A3 and B2. In order to understand activity and specificity of these plant defensins, the structure of a protein belonging to subfamily A2 is needed. We report the three-dimensional solution structure of Ah-AMP1 as determined from twodimensional 1 H nuclear magnetic resonance data. The structure features all the characteristics of the ''cysteine-stabilized ␣␤-motif.'' A comparison of the structure, the electrostatic potential surface and regions important for interaction with the fungal receptor, is made with Rs-AFP1 (plant defensin of subfamily A3). Thus, residues important for activity and specificity have been assigned. Proteins 1999;37:388-403.

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Expression of functional Raphanus sativus antifungal protein in yeast

Cited by 29 publications

References 12 publications

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae

Mutational Analysis of a Plant Defensin from Radish (Raphanus sativus L.) Reveals Two Adjacent Sites Important for Antifungal Activity

The three-dimensional solution structure ofAesculus hippocastanum antimicrobial protein 1 determined by1H nuclear magnetic resonance

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