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.     

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.     

Isolation of lectin and albumin from Pisum sativum var. macrocarpon ser. cv. sugar snap

A mannose- and glucose-binding lectin bearing considerable sequence similarity to other legume lectins was isolated using a simple procedure, from legumes of the sugar snap Pisum sativum var. macrocarpon. The lectin was unadsorbed on Affi-gel blue gel and Q-Sepharose in 10 mM Tris-HCl buffer (pH 7.2) and adsorbed on SP-Toyopearl in 50 mM NaOAc buffer (pH 5). An albumin could also be purified at the same time. It was unadsorbed on Affi-gel Blue gel, adsorbed on Q-Sepharose and unadsorbed on SP-Toyopearl under the aforementioned chromatographic conditions. The lectin was almost identical in N-terminal sequences of its alpha- and beta-subunit to lectin from P. sativum L. var. Feltham First except for the 19th N-terminal residue of the beta-subunit. The lectin was devoid of antifungal activity. Out of the 15 N-terminal amino acids examined in pea albumin, three were different between the two varieties of P. sativum.     

Characterization of two novel defense peptides from pea (Pisum sativum) seeds

A fraction that possesses antifungal activity against Aspergillus niger has been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75. On further purification by reverse-phase high performance liquid chromatography, two small cysteine-rich polypeptides were obtained (Psd1 and Psd2). They are localized primarily in vascular bundles and epidermis tissues of pea pods and exhibit high antifungal activity toward several fungi, displaying IC(50) values ranging from 0.04 to 22 microg/ml. This inhibitory activity decreases when A. niger growth medium is supplemented with cations such as Ca(2+), Mg(2+), Na(+), and K(+). Although the primary sequence of both Psd1 and Psd2 shows homology with other plant defensins, they cannot easily be assigned to any established group.     

Isolation of alliumin, a novel protein with antimicrobial and antiproliferative activities from multiple-cloved garlic bulbs

A protein designated alliumin, with a molecular mass of 13 kDa and an N-terminal sequence similar to a partial sequence of glucanase, and demonstrating antifungal activity against Mycosphaerella arachidicola, but not against Fusarium oxysporum, was isolated from multiple-cloved garlic (Allium sativum) bulbs. The protein, designated as alliumin, was purified using ion exchange chromatography on DEAE-cellulose, CM-cellulose and Mono S, affinity chromatography on Affi-gel blue gel, and gel filtration on Superdex 75. Alliumin was unadsorbed on DEAE-cellulose, but was adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. Its antifungal activity was retained after boiling for 1 h and also after treatment with trypsin or chymotrypsin (1:1, w/w) for 30 min at room temperature. Alliumin was inhibitory to the bacterium Pseudomonas fluorescens and exerted antiproliferative activity toward leukemia L1210 cells. However, it was devoid of ribonuclease activity, protease activity, mitogenic activity toward mouse splenocytes, and antiproliferative activity toward hepatoma Hep G2 cells.     

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.     

Protein and peptide factors from Bacillus sp.739 inhibit the growth of phytopathogenic fungi

Thermolabile peptides inhibiting the growth of Helminthosporium sativum, a facultative phytopathogen, have been isolated from the low-molecular-weight fraction of extracellular metabolites of the strain Bacillus sp. 739. Paper chromatography of the fraction, followed by bioautography, revealed the presence of three components exhibiting antifungal activity. These components were separated by gel chromatography on Toyopearl HW-40. SDS-PAGE (the Laemmli procedure) demonstrated that only one component was a protein (MW, approximately 14 kDa). The other two substances were polypeptides with molecular weights less than 6 kDa each. The protein factor inhibited the growth of H. sativum with a minimum effective concentration of 0.1 to 0.2 mg/ml.     

Purification and subunit structure of RNA polymerase II from the pea.

DNA-dependent RNA polymerase II (EC 2.7.7.6) from pea seedlings (Pisum sativum var. Alaska) has been purified to homogeneity, as judged by native polyacrylamide electrophoresis. The procedure includes polyethyleneimine precipitation and elution, ammonium sulfate precipitation, DEAE-Sephadex chromatography, phosphocellulose chromatography, and heparin-Sepharose chromatography. The enzyme purified almost to homogeneity has a specific activity of 200 nmol/mg per 15 min at 30 degrees C with denatured calf thymus DNA as template. The enzyme activity is 50% inhibited in the presence of 0.05 migrograms/ml of alpha-amanitin. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that pea RNA polymerase II is composed of eight subunits with molecular weights and molar ratios (in parentheses) of 170 000 (0.9), 140 000 (1.0), 43 000 (1.5), 26 000 (2.0), 22 500 (1.2), 21 500 (0.6), 18 500 (1.6) and 17 500 (2.3). The structure is closely similar to that of cauliflower RNA polymerase II.     

Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit

The taste-modifying protein, miraculin, has the unusual property of modifying a sour taste into a sweet taste. Previous attempts to isolate miraculin from deeply colored alkaline extracts of the miracle fruit were unsuccessful. We found that miraculin is extracted with 0.5 M NaCl solution. The extracted solution is colorless and shows the strong sweet-inducing activity. Miraculin was purified from the extracted solution by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose affinity chromatography. The purified miraculin thus obtained gave a single sharp peak in reverse phase high performance liquid chromatography, indicating that it is highly pure. The sample also gave a single band having molecular weight 28,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was much lower than the values reported previously (40,000-48,000). The amino acid composition of the purified miraculin was determined. Sequence analysis of the purified miraculin indicated that it is composed of a pure single polypeptide and identified 20 amino-terminal amino acids. The purified miraculin contained as much as 13.9% of sugars, which consisted of glucosamine, mannose, galactose, xylose, and fucose in a molar ratio of 3.03:3.00:0.69:0.96:2.12.     

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

Plant defensins are small cysteine-rich proteins that present high activity against fungi and bacteria and inhibition of insect proteases and alpha-amylases. Here, we present the expression in Pichia pastoris, purification and characterization of the recombinant Pisum sativum defensin 1(rPsd1); a pea defensin which presents four disulfide bridges and high antifungal activity. For this, we had to overcome the inefficiency of the STE13 protease. Our strategy was to clone the corresponding cDNA directly in-frame with a variant of the widely used secretion signal from the Saccharomyces cerevisiae alpha-mating factor, devoid of the STE13 proteolytic signal cleavage sequence. Using an optimized expression protocol, which included a buffered basal salt media formulation, it was possible to obtain about 63.0mg/L of 15N-labeled and unlabeled rPsd1. The recombinants were purified to homogeneity by gel filtration chromatography, followed by reversed-phase HPLC. Mass spectrometry of native and recombinant Psd1 revealed that the protein expressed heterologously was post-translationally processed to the same mature protein as the native one. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein had the same folding when compared to native Psd1. In addition, the rPsd1 was fully active against Aspergillus niger, if compared with native Psd1. To our knowledge, this is the first heterologous expression of a fully active plant defensin in a high-yield flask.     

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.