Passiflin,a novel dimeric antifungal protein from seeds of the passion fruit

The intent was to isolate an antifungal protein from seeds of the passion fruit (Passiflora edulis) and to compare its characteristics with other antifungal proteins and bovine β-lactoglobulin in view of its N-terminal amino acid sequence similarity to β-lactoglobulin. The isolation procedure entailed ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on DEAE-cellulose, and FPLC-gel filtration on Superdex 75. The isolated 67-kDa protein, designated as passiflin, exhibited an N-terminal amino acid sequence closely resembling that of bovine β-lactoglobulin. It is the first antifungal protein found to have a β-lactoglobulin-like N-terminal sequence. Its dimeric nature is rarely found in antifungal proteins. It impeded mycelial growth in Rhizotonia solani with an IC₅₀ of 16 μM and potently inhibited proliferation of MCF-7 breast cancer cells with an IC₅₀ of 15 μM. There was no cross-reactivity of passiflin with anti-β-lactoglobulin antiserum. Intact β-lactoglobulin lacks antifungal and antiproliferative activities and is much smaller in molecular size than passiflin. However, it has been reported that hydrolyzed β-lactoglobulin shows antifungal activity. The data suggest that passiflin is distinct from β-lactoglobulin.     

Mungin, a novel cyclophilin-like antifungal protein from the mung bean

A protein designated mungin, isolated from mung bean (Phaseolus mungo) seeds, possessed activity against the fungi Rhizoctonia solani, Coprinus comatus, Mycosphaerella arachidicola, Botrytis cinerea, and Fusarium oxysporum. The 18-kDa protein also possessed a novel N-terminal sequence with similarity to cyclophilins. It exerts an inhibitory action against alpha- and beta-glucosidases suppresses [(3)H]thymidine in corporation by mouse splenocytes.     

Pr-1, a novel antifungal protein from pumpkin rinds

A novel antifungal protein, M_r = ca. 40 kDa, was isolated from pumpkin rind and designated Pr-1. When purified by anion exchange chromatography and HPLC, it inhibited growth of several fungi including Botrytis cinerea, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani, as well as the yeast, Candida albicans, at 10–20 μM. It did not inhibit growth of Escherichia coli or Staphylococcus aureus even at 200 μM. Laser scanning microscopy of fungal cells exposed to rhodamine-labeled Pr-1 revealed that the protein accumulated and was localized on the cell surface. Uptake of the vital stain, SYTOX Green, was enhanced when fungal conidia were treated with Pr-1 suggesting that the protein has membrane permeabilization activity. Pr-1 was thermostable at 70°C and did not lyse human red blood cells at 128 μM suggesting that the protein may be useful as an antifungal agent with little, if any human cytotoxicity.     

Actinidic acid, a new triterpene phytoalexin from unripe kiwi fruit

Seven phytoalexins (1-7), including a new compound, were isolated from the peel of unripe kiwi fruit (Actinidia deliciosa cv. Golden King) that had been wounded and inoculated with Colletotrichum musae. The new phytoalexin (1) was identified as 2alpha,3beta,23-trihydroxy-12,20(30)-ursadien-28-oic acid, and named actinidic acid. Phytoalexins 2-6 are known triterpenes but have not previously been described as phytoalexins. Phytoalexin 7 is the same triterpene as the phytoalexin of nectarine fruit.     

Pectin methylesterase inhibitor cDNA from kiwi fruit

We have newly isolated one partial pectin methylesterase inhibitor (PMEI) and two full-length cDNA clones from a kiwi fruit cDNA library. The two full-length cDNA clones, Adpmei-1 and Adpmei-2, had an open reading frame of 185 amino acids, including a predicted signal peptide sequence necessary for localization in the cell-wall space. As the deduced amino acid sequence of the cloned fragment was almost same as the sequence of the previously purified PMEI protein (Camardella et al., Eur J Biochem 267:4561–4565), the clones were considered to be cDNAs encoding PMEI protein. Southern blot analysis indicated a low-copy number of the PMEI genes. Transgenic analysis of asparagus calli expressing a kiwi fruit PMEI gene driven by the CaMV 35S promoter demonstrated in vivo inhibition effects of PMEI on the endogenous pectin methylesterase (PME) activity. The relative expression levels of the PMEI genes in kiwi fruit, analyzed by competitive PCR, increased with the progression of fruit maturation. Given that PME activity also showed its highest level at the fully ripened stage of maturation, the increase in PMEI expression may not indicate direct inhibitory effects on the PME activity and fruit maturation process.     

Purification of allivin,a novel antifungal protein from bulbs of the round-cloved garlic

A novel antifungal protein, designated allivin, was isolated from bulbs of the round-cloved garlic Allium sativum var. round clove with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and FPLC-gel filtration on Superdex 75. Allivin possessed an N-terminal sequence demonstrating very little similarity to sequences of Allium sativum chitinases and ribosome inactivating proteins. Allivin exhibited a molecular weight of 13 kDa in gel filtration and SDS-polyacrylamide gel electrophoresis. It displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola. It inhibited translation in a cell-free rabbit reticulocyte system with an IC50 of 1.6 microM.     

Hispin,a novel ribosome inactivating protein with antifungal activity from hairy melon seeds

A ribosome inactivating protein demonstrating a molecular mass of 21 kDa and a novel N-terminal sequence was isolated from seeds of the hairy melon. The purification procedure involved affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose and Mono S. The protein designated hispin inhibited translation in the cell-free rabbit reticulocyte lysate system with an IC50 of 165 pM and exhibited N-glycosidase activity. Antifungal activity was also observed.     

Cicerarin,a novel antifungal peptide from the green chickpea

A peptide designated cicerarin, with an N-terminal amino acid sequence VKSTGRADDDLAVKTKYLPP dissimilar from known proteins and peptides and a molecular mass of 8kDa, was isolated from seeds of the green chickpea Cicer arietinum cv green chickpea. Cicerarin was isolated with a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10mM Tris-HCl buffer (pH 7.3). Cicerarin exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100 degrees C for 15min.     

Alveolarin, a novel antifungal polypeptide from the wild mushroom Polyporus alveolaris

An antifungal polypeptide, with a molecular mass of 28 kDa as judged by gel filtration and appearing as a single band with a molecular mass of 14 kDa in sodium dodecyl suflate-polyacrylamide gel electrophoresis, was isolated from fresh fruiting bodies of the mushroom Polyporus alveolaris. The antifungal polypeptide, designated as alveolarin, demonstrated an inhibitory action on mycelial growth in Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. Alveolarin was isolated with a procedure that entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75 by fast protein liquid chromatography.     

Strawberry fruit protein with a novel indole-acyl modification

Achenes and receptacle tissue of Fragaria vesca, L. cultivar Yellow Wonder were shown to contain conjugated indole-3-acetic acid (IAA) that was not soluble in organic solvents and yielded IAA after strong alkaline hydrolysis, suggestive of IAA attached to plant proteins. This solvent insoluble conjugated IAA accounted for between 0.4 and 4 ng of IAA per gram fresh weight of tissue in both achenes and receptacles. To investigate this strawberry conjugate class further, a polyclonal antibody was produced to IAA–glycine attached to BSA that detected neutral indole acid esters, monocarboxylic-amino acid IAA conjugates and IAA proteins. Using immunoblotting, both achenes and receptacles of strawberry were shown to have primarily an immuno-detectable band at 76 kDa. Two-dimensional polyacrylamide gel electrophoresis yielded a wide band that was analyzed by LC–MS/MS analysis following in-gel trypsin digestion. Peptides derived from the immuno-detectable band were tentatively identified by peptide fragment analysis as being from either a chaperonin related to the hsp60 class of proteins or, alternatively, an ATP synthase. This is one of the first reports of an IAA modified protein in fruit tissue.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Isolation of pisumin,a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var macrocarpon

An antifungal protein with a novel N-terminal sequence GVGAAYGCFG and a molecular mass of 31 kDa was isolated from the legumes of the sugar snap pea Pisum sativum var. macrocarpon. The protein, designated pisumin, exhibited antifungal activity against Coprinus comatus and Pleurotus ostreatus and much weaker activity against Fusarium oxysporum and Rhizoctonia solani. Pisumin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 6 microM. Pisumin was similar to other leguminous antifungal proteins in that it was adsorbed on Affi-gel blue gel and CM-Sepharose.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.