Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

P-糖蛋白的表达及其结合肽的筛选

为获得P 糖蛋白胞外段 ,构建了高效表达载体pGEX Pgp ,转化大肠杆菌DH5α ,进行表达、鉴定及纯化 ,以获得的融合蛋白为靶蛋白 ,筛选噬菌体随机 12肽库 ,免疫细胞化学方法进行鉴定 .SDS PAGE分析 ,表达出约 30kD大小的蛋白 ;从噬菌体随机肽库中筛选获得了与P 糖蛋白特异性结合的噬菌体阳性克隆 ,测序获得了其特异性结合肽序列 :NDGLLFTWQPSP .免疫细胞化学结果显示 :筛选得到的噬菌体阳性克隆可与耐药细胞BIU 87 ADM结合 ,而与敏感细胞BIU 87不结合 .结果表明 ,筛选获得的结合肽可与耐药的肿瘤细胞结合 ,表现出一定的肿瘤特异性 .P 糖蛋白结合肽的筛选 ,为进行人膀胱癌多药耐药的靶向治疗等工作奠定了基础 .     

苦瓜籽核糖体失活蛋白的理化性质及生物活性

采用硫酸铵分级分离,假配基亲和层析和ＳｅｐｈａｃｒｙｌＳ－１００分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白（ＲＩＰ）．经ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ、ＩＥＦ和ＰＡＳ方法分析均表明为单一蛋白着色带或单一糖蛋白着色带．根据ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ Ｇ－１５０分子筛层析结果计算其相对分子质量为３．０×１０４,经ＩＥＦ－ＰＡＧＥ结果计算其ｐＩ为８．９～９．０．对无细胞系统中蛋白质生物合成抑制活性明显,其ＩＣ５０为５．３×１０－ １０ ｍ ｏｌ／Ｌ左右．体外生物活性试验结果表明其对人肝癌细胞、Ｖｅｒｏ、ＳＰ２／０、３Ｔ３、Ｋｂ、Ｎａｖａｎａ 等肿瘤细胞株均表现有不同程度的抑制作用．而对完整细胞人胚肺二倍体细胞却毒性极小．因此,上述实验结果为该ＲＩＰ的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础．     

Fungal elicitor protein PebC1 from Botrytis cinerea improves disease resistance in Arabidopsis thaliana

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml^?1 activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway.     

Molecular cloning and recombinant expression of cytochrome P450 CYP6B6 from Helicoverpa armigera in Escherichia coli

The cytochrome P450 s play a significant role in the detoxification of plant allelochemicals and synthetic insecticides in Lepidoptera. In the cotton bollworm Helicoverpa armigera, 2-tridecanone and quercetin can induce P450-dependent monooxygenase activity increased, to further the characterization of P450, the CYP6B6 of cotton bollworm (H. armigera) was cloned, sequenced and expressed in pMAL-p2x vector and expressed in Escherichia coli. The deduced amino acid sequences of cytochrome P450 in the midgut and fat body of H. armigera showed 98.23 and 97.84 % similarity with CYP6B6, respectively. According to nomenclature of P450 s, the P450 genes we got belong to CYP6B. Purification of recombinant protein based on the affinity of MBP for maltose was achieved by Mal-Tag magnetic beads. The purified protein was used to raise polyclonal antibody according to classical procedure. SDS–PAGE and Western blot results indicated that MBP-CYP6B6 had been successfully expressed. The ethoxycoumarin-O-deethylase activity of the purified recombinant protein was 36.5 ± 8.12 pmol of 7-hydroxycoumarin/min/mg protein, which showed the fusion MBP-CYP6B6 had the ability to o-deethylase of 7-ethoxycoumarin.     

The purification and characterization of a novel alkali-stable pectate lyase produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> PB1

Pectinase is an important kind of enzyme with many industrial applications, among which pectinases produced by bacteria were scarce compared with fungal sources. In this study, a novel bacterium which produced extracellular pectinase was firstly isolated from flue-cured tobacco leaves and identified as Bacillus subtilis PB1 according to its 16S rRNA gene. The pectinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography, after which molecular weight was determined as 43.1?±?0.5 kDa by SDS–PAGE. Peptide mass fingerprinting of the pectinase by MALDI-TOF MS showed that the purified enzyme shared homology with pectate lyase and was designated as BsPel-PB1. The optimal temperature for BsPel-PB1 was 50 °C. The optimal pH was pH 9.5 for BsPel-PB1 while it had a broad pH stability from 5 to 11. The values of K _m and V _max were 0.312 mg/mL and 1248 U/mL, respectively. Accordingly, the BsPel-PB1 was a novel alkaline pectate lyase which could find potential application as a commercial candidate in the pectinolytic related industries.     

Purification and characterization of elicitor protein from Phytophthora colocasiae and basic resistance in Colocasia esculenta

An elicitor was identified in the fungus Phytophthora colocasiae. The molecular weight of the purified elicitor was estimated by means of gel filtration chromatography and SDS-PAGE and was estimated as 15kDa. Protease treatment severely reduced its activity, allowing the conclusion that the elicitor is proteinaceous. Infiltration of a few nanograms of this proteinaceous elicitor into taro leaves caused the formation of lesions that closely resemble hypersensitive response lesions. The elicitation of the cells was effective in the induction of the activity of lipoxygenase. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, after the infiltration of the elicitor protein. After few days, systemic acquired resistance was also induced. Thus, taro plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence. The obtained results give important information regarding the plant-pathogen interactions, mainly as subsidy for taro improvement against Phytophthora leaf blight.     

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.     

Purification and partial characterization of a novel hemagglutinating glycoprotein from the cultured mycelia of Hericium erinaceus

HEG-5, a novel glycoprotein with hemagglutinating activity, was firstly isolated and purified from the cultured mycelia of Hericium erinaceus CZ-2. SDS–PAGE, Native-PAGE and MALDI-TOF-MS proved that HEG-5 was a single band with the molecular weight of approximately 14.4 kDa. HEG-5 had the protein: polysaccharide ratio of approximately 10:1 (%/%) and contained d-glucose, l-rhamnose, d-galactose and d-mannose with a molar ratio of 1.00:1.09:2.45:7.14 in polysaccharide fraction. HEG-5 was an acidic glycoprotein with a PI value of 6.3 and the higher content of acidic amino acids (Asp, 12.42 ± 0.25% and Glu, 12.24 ± 0.26%) in protein fraction. FT-IR and NMR spectra revealed that HEG-5 contained the protein and carbohydrate portions with (1→4)-linked β-galactose residues and β-linked glucose residues. Circular dichroism (CD) demonstrated that HEG-5 was a β-sheet predominant glycoprotein. Hemagglutination assay proved it was a thermo-unstable glycoprotein. The HEG-5 structural novelty was finally presented by protein sequencing and modeling by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODEL Workspace service.     

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor-mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat-stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor-encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide-associated complex domain. The peaT1 gene was cloned into the expression vector pET-28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.     

Characterization of a new antifungal lipid transfer protein from wheat

Lipid transfer proteins (LTPs) are members of the family of pathogenesis-related proteins (PR-14) that are believed to be involved in plant defense responses. In this study, a novel gene Ltp 3F1 encoding an antifungal protein from wheat (Sumai 3) was subcloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation followed by gel permeation chromatography. Molecular phylogeny analyses of wheat Ltp 3F1 gene showed a strong identity to other plant LTPs. Predicted three-dimensional structural model showed the presence of 6 α-helices and 9 loop turns. The active site catalytic residues Gly30, Pro50, Ala52 and Cys55 may be suggested for catalyzing the reaction involved in lipid binding. SDS–PAGE analysis confirmed the production of recombinant fusion protein. The LTP fusion protein exhibited a broad-spectrum antifungal activity against Alternaria sp., Rhizoctonia solani, Curvularia lunata, Bipolaris oryzae, Cylindrocladium scoparium, Botrytis cinerea and Sarocladium oryzae. Gene cassette with cyanamide hydratase (cah) marker and Ltp 3F1 gene was constructed for genetic transformation in tobacco. Efficient regeneration was achieved in selective media amended with cyanamide. Transgenic plants with normal phenotype were obtained. Results of PCR and Southern, Northern and Western hybridization analyses confirmed the integration and expression of genes in transgenic plants. Experiments with detached leaves from transgenic tobacco expressing Ltp 3F1 gene showed fungal resistance. Due to the innate potential of broad-spectrum antifungal activity, wheat Ltp 3F1 gene can be used to enhance resistance against fungi in crop plants.     

Delta 6-desaturase activity in liver microsomes of rats fed diets enriched with cholesterol and/or omega 3 fatty acids.

A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.     

Molybdophyllysin,a toxic metalloendopeptidase from the tropical toadstool,Chlorophyllum molybdites

A toxic protein, dubbed molybdophyllysin, was isolated from the tropical toadstool Chlorophyllum molybdites by following its lethal effect in mice. Analysis of the protein using SDS–PAGE revealed a single 23-kDa band. Sequence analysis of molybdophyllysin tryptic fragments showed that this protein is highly homologous to metalloendopeptidases (MEPs) obtained from edible mushrooms, such as Grifola frondosa, Pleurotus ostreatus, and Armillaria mellea. These proteins include a HEXXH+D zinc-binding motif known as aspzincin. Accordingly, molybdophyllysin is a member of the deuterolysin family of zinc proteases. Molybdophyllysin retained its proteolytic activity at temperatures up to 60 °C with an optimum pH of 7.0. The activity was inhibited by both 1,10-phenanthroline and N-bromosuccinimide, but molybdophyllysin exhibited strong resistance to SDS.     

A novel elicitor identified from Magnaporthe oryzae triggers defense responses in tobacco and rice

Key message

Our studies indicate a potential important elicitor candidate which can aid in the fight against a worldwide disease, rice blast.

Abstract

In this study, we report the purification, identification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip2) secreted from an important pathogenic fungus, Magnaporthe oryzae. The protein fraction was isolated from the culture filtrate of M. oryzae and identified by de novo sequencing. The elicitor-encoding gene mohrip2 was cloned following sequence comparison and PCR amplification. This 459-bp gene encodes a 152-residue polypeptide that contains an 18-residue signal peptide and exhibits a pI of 4.72 and an apparent molecular mass of 16 kDa. The hypothetical protein, MoHrip2, was expressed in Escherichia coli, and both the recombinant and the endogenous protein caused necrotic lesions in tobacco leaves. In addition to phenolic compound deposition and alkalization of the extracellular medium, MoHrip2 also induced hydrogen peroxide production and nitric oxide accumulation in tobacco cells. Moreover, rice seedlings treated with MoHrip2 exhibited pronounced resistance to M. oryzae compared with control seedlings.     

Purification of NADH: Hypothiocyanite oxidoreductase inStreptococcus sanguis

NADH: hypothiocyanite oxidoreductase (NHOR) activity, found in some oralStreptococci,is postulated to protect these microorganisms against salivary peroxidase-produced hypothiocyanite. NHOR, however, has not been purified so far. The purification of NHOR from crude extracts ofStreptococcus sanguisNCTC 7863 strain (by ultrafiltation and anion-exchange chromatography) revealed one fraction of 125 ± 1 kDa. However, SDS–PAGE electrophoresis provided a single protein of 21.1 ± 1.2 kDa. This last discovery suggests that NHOR enzyme is a hexameric complex having six subunits.     

Overexpression of MsALMT1, from the Aluminum-Sensitive Medicago sativa, Enhances Malate Exudation and Aluminum Resistance in Tobacco

Aluminum (Al) toxicity is a major limiting factor for plant growth and crop production in acidic soils. Al-induced organic acid (OA) exudation plays an important role in plant Al resistance. The exudation of OAs is mediated by membrane-localized OA transporters. In our previous study, a gene encoding the Al-induced malate transporter (MsALMT1) was identified in the roots of the Al-sensitive plant Medicago sativa L. cv. Yumu no. 1 (YM1). To further validate the function of MsALMT1, transgenic plants that overexpressed MsALMT1 under the control of the CaMV 35S (35S) promoter were generated. This transgenic tobacco showed an enhanced capacity for malate efflux and better Al resistance than wild type (WT) plants after exposure to 30 μM Al for 24 h. The Al content in the transgenic plant roots decreased to 40–52 % of that in WT plant roots. These results demonstrate that MsALMT1 is an Al-resistant gene in YM1 and encodes a malate transporter, the overexpression of which effectively enhances the Al resistance of transgenic tobacco plants.     

A study on the allergenicity and ontogeny of Acacia farnesiana pollen grains in guinea pigs

Pollen grains as the angiosperm male gametophytes transfer male genetic material during sexual reproduction. Pollen grains are among the important plant allergens, such that almost 80–90 % of plant allergens are of pollen origin. Acacia farnesiana is a plant with economical values, and due to its resistance to dry climates, it has had a widespread distribution in Southern Iran. This study was aimed at investigating the allergenicity and the ontogeny of pollen grains of A. farnesiana. Pollen grains were collected from the blossoms and flowers of A. farnesiana in the suburbs of Ahvaz-Iran. Pollen extracts (15 %) were prepared in PBS (pH 7.2). 4–6-week-old male guinea pigs (Hartly) were used for allergenicity tests. Skin tests showed a significant increase in flare diameter. Clinical tests also showed a significant change in the levels of eosinophils, neutrophils, and IgE. Histotechnical analysis was performed on male gametophytes, and photographs were taken using a camera-equipped light microscope. Pollen characteristics were identified using both light and electron microscopy. Sporoderm ultrastructure and pollen morphology were studied using Scanning Electron Microscopy. PAGE analysis of the total protein content of mature pollen grains showed seven clear bands of 10–83 kDa.     

Polyamine oxidase from water hyacinth: purification and properties

Polyamine oxidase was purified to homogeneity from leaves of water hyacinth by the criterion of sodium dodecyl sulfate gel electrophoresis (SDS disc PAGE). The enzyme showed a high specificity for spermidine and spermine (K_m values 28 micromolar and 20 micromolar, respectively). The optimal pH of the enzyme for both spermidine and spermine was 6.5. The molecular weight of the enzyme estimated by Sephadex G-200 gel filtration was 87,000, while SDS disc PAGE gave a single band at the molecular weight of 60,000. Octamethylenediamine and quinacrine were strong inhibitors of the enzyme, but p-chloromercuribenzoate was without effect. A prosthetic group in the enzyme was identified as flavin adenine dinucleotide.     

Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His₆-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652 IU/L after 2 days of cultivation in the induction medium. The His₆-tagged rNC-CDH1 was purified through a one-step Ni–NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 7451 IU/L (0.89 mg protein/mL), with a specific CDH activity of 8.37 IU/mg. The purity of the enzyme was examined by SDS–PAGE, and a single band corresponding to a molecular weight of about 120 kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25–70 °C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K_m value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k_cat remains relatively constant when the chain length changes.