Band In SDS-PAGE Research Articles

Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, Clarias batrachus (L.).

Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E2)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100ngml-1 for Vg1 and 40ngml-1 for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E2 induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.

English

Bovine herpesvirus-1 (BHV-1) causes Infectious bovine rhinotracheitis/Pustular vulvovaginitis in cattle. Glycoprotein D (gD) of BHV-1 represents a major component of the viral envelope and is a dominant immunogen. gD encoding gene was expressed in baculovirus-insect cell system. Viral genomic DNA extracted from BHV-1 grown on Madin-Darby Bovine Kidney (MDBK) cell monolayer was used as a template for PCR amplification of gD gene (1255 bp). Gel purified gD gene was used for directional cloning into pENTR/SD/D Directional TOPO vector to produce entry clone. Recombinant plasmids were screened by PCR and restriction enzyme (RE) digestion for gD gene insert.  Endotoxin free purified plasmids were then subjected to LR recombination reaction with baculovirus linear DNA. LR recombination mix was transfected into Sf-9 cells and observed for appearance of cytopathic effects (CPE). Recombinant virus was serially passaged for 3 more generations and the 4th passage viral stock was used to infect fresh Sf-9 cells for gene expression study. Recombinant gD protein was immunoprecipitated and when subjected to SDS-PAGE and western blot analysis protein band of ~70 kDa was detected consistently. The recombinant gD protein was further weakly confirmed by dot-ELISA indicating its limited potential as a coating antigen in gD-based diagnostic ELISA. Key words: Bovine herpesvirus-1 (BHV-1), Madin-Darby Bovine Kidney (MDBK) cells, Sf-9 cells, pENTR TOPO vector, Baculovirus mediated gene expression, glycoprotein D, eukaryotic expression,

Extraction and purification of protease inhibitor(s) from seeds of Helianthus annuus with effects on Leptinotarsa decemlineata digestive cysteine protease

Abstract The aim of the current investigation was to purify cysteine protease inhibitors from sunflower ( Helianthus annuus L. cv. Record) (Asteraceae) seeds with potential activity on gut protease of Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Ammonium sulfate precipitated proteinaceous fractions; 0–30%, 30–50%, 50–70%, and 70–100% showed 41.03%, 48.66%, 41.53%, and 28.83% inhibition on the last instar larval gut general protease activity, respectively. Also, fraction 30–50% showed the highest inhibitory effect on digestive general protease activity of all developmental stages. This fraction was purified using two different chromatography techniques; ion-exchange using DEAE and affinity using SiO 2 -CPB larval gut enzyme mix. Four peaks of protein were eluted from ion-exchange chromatography using NaCl step gradient. When used Z-Ala-Arg-Arg-4msNA as cysteine protease substrates, the purification fold of first fraction was obtained 28.31, also the yield was 66.89%. Fraction of affinity chromatography obtained 26.98 purification fold and yielded 53.96%. Both of the purified protein fractions from two methods showed two similar bands in SDS-PAGE with apparent molecular mass of 14 and 15 kDa. Consequently, the high level of purification fold and yield suggest that both methods were appropriate for purification, but affinity was more specific because CPB gut enzyme was used as ligand.

An Amyloid-Like Pathological Conformation of TDP-43 Is Stabilized by Hypercooperative Hydrogen Bonds.

TDP-43 is an essential RNA-binding protein forming aggregates in almost all cases of sporadic amyotrophic lateral sclerosis (ALS) and many cases of frontotemporal lobar dementia (FTLD) and other neurodegenerative diseases. TDP-43 consists of a folded N-terminal domain with a singular structure, two RRM RNA-binding domains, and a long disordered C-terminal region which plays roles in functional RNA regulatory assemblies as well as pernicious aggregation. Evidence from pathological mutations and seeding experiments strongly suggest that TDP-43 aggregates are pathologically relevant through toxic gain-of-harmful-function and/or harmful loss-of-native-function mechanisms. Recent, but not early, microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates in vitro and in cells strongly suggest that TDP-43 aggregates have a self-templating, amyloid-like structure. Based on the importance of the Gln/Asn-rich 341–367 residue segment for efficient aggregation of endogenous TDP-43 when presented as a 12X-repeat and extensive spectroscopic and computational experiments, we recently proposed that this segment adopts a beta-hairpin structure that assembles in a parallel with a beta-turn configuration to form an amyloid-like structure. Here, we propose that this conformer is stabilized by an especially strong class of hypercooperative hydrogen bonding unique to Gln and Asn sidechains. The clinical existence of this conformer is supported by very recent LC-MS/MS characterization of TDP-43 from ex vivo aggregates, which show that residues 341–367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation. Its distinct pattern of SDS-PAGE bands allows us to link this conformer to the exceptionally stable seed of the Type A TDP-43 proteinopathy.

Effect of ozone gas processing on physical and chemical properties of wheat proteins

Purpose: To investigate the effects of ozone treatment on chemical and physical properties of wheat ( Triticum aestivum L.) gluten, glutenin and gliadin. Methods: Wheat proteins isolated from wheat flour were treated with ozone gas. The physical and chemical properties of gluten proteins were investigated after treatment with ozone gas, with 5 g/h produced as a function of time (0, 30, and 60 min) in the study. To check whether the process of ozonation promoted changes in the quality of gluten proteins, sulfhydryl groups (SH), differential scanning calorimetry (DSC), secondary structure, SDS-PAGE, and rheology analyses were performed. Results: Sulfhydryl group contents of wheat proteins ranged from 1.1 to 7.12 μmol/g. Sulfhydryl group content for all ozonated proteins was significantly lower than that of the control samples. Gluten proteins showed reduced SDS-PAGE band intensities of both high (HMW) gluten and glutenin subunits with increasing ozone gas treatment. The denaturation temperatures (Td) of ozonated gluten proteins were higher (99.80–106.79 °C) and the enthalpies of the ozonated gluten proteins were lower than those of the control samples. The storage moduli (G') and loss moduli (G”) of gluten and glutenin tended to increase from 7.84 to 10.20 KPa and 43.19 to 48.28 KPa, and from 3.33 to 4.06 KPa and 20.74 to 22.56 KPa, respectively, as ozone exposure increased from 0 to 30 min. Conclusion: Ozone gas can oxidize wheat proteins. Exposing wheat proteins to ozone gas for an extended time (60 min) deteriorated wheat protein quality. Keywords: Ozone treatment, Gluten proteins, Chemical changes, Rheological studies, CD spectroscopy, Thermal properties

Purification and characterization of novel fibrin(ogen)olytic protease from Curcuma aromatica Salisb.: Role in hemostasis

BackgroundThe proteases from turmeric species have procoagulant and fibrinogenolytic activity. This provides a scientific basis for traditional use of turmeric to stop bleeding and promote wound healing processes. PurposeOur previous studies revealed that fibrinogenolytic action of crude enzyme fraction of Curcuma aromatica Salisb., was found to be more influential than those of Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. Hence, the purpose of this study is to purify and characterize protease from C. aromatica and to explore its role in wound healing process. MethodsThe protease was purified by Sephadex G-50 gel permeation chromatography. Peak with potent proteolytic activity was subjected to rechromatography and then checked for homogeneity by SDS-PAGE and native PAGE. Furthermore purity of the peak was assessed by RP-HPLC and MALDI-TOF. The biochemical properties, type of protease, kinetic studies, fibrinogenolytic, coagulant and fibrinolytic activities were carried out. ResultsThe two proteolytic peaks were fractionated in gel permeation chromatography. Among these, the peak-II showed potent proteolytic activity with specific activity of 10units/mg/min and named as C. aromatica protease-II (CAP-II). This protein resolved into a single sharp band both in SDS-PAGE (reducing and non-reducing) as well as in native (acidic) PAGE. It is a monomeric protein, showing sharp peak in RP-HPLC and its relative molecular mass was found to be 12.378kDa. The caseinolytic and fibrinolytic activity of CAP-II was completely inhibited by phenylmethylsulfonylfluoride (PMSF). The CAP-II exhibited optimum temperature of 45°C and optimum pH of 7.5. The Km and Vmax of CAP-II was found to be 1.616µg and 1.62units/mg/min respectively. The CAP-II showed hydrolysis of all three subunits of fibrinogen in the order Aα>Bß>γ. The CAP-II exhibited strong procoagulant activity by reducing the human plasma clotting time. It also showed fibrinolytic activity by complete hydrolysis of α-polymer and γ-γ dimer present in fibrin. ConclusionThe CAP-II is a novel serine protease from C. aromatica, which has been demonstrated to stop bleeding and initiate wound healing through its procoagulant and fibrin(ogen)olytic activities. Our study demonstrates the possible role of CAP-II, as therapeutic enzyme to stop bleeding at the time of wounding.

Structural proteins of Enterococcus faecalis bacteriophage ϕEf11.

ABSTRACTϕEf11, a temperate Siphoviridae bacteriophage, was isolated by induction from a root canal isolate of Enterococcus faecalis. Sequence analysis suggested that the ϕEf11 genome included a contiguous 8 gene module whose function was related to head structure assembly and another module of 10 contiguous genes whose products were responsible for tail structure assembly. SDS-PAGE analysis of virions of a ϕEf11 derivative revealed 11 well-resolved protein bands. To unify the deduced functional gene assignments emanating from the DNA sequence data, with the structural protein analysis of the purified virus, 6 of the SDS-PAGE bands were subjected to mass spectrometry analysis. 5 of the 6 protein bands analyzed by mass spectrometry displayed identical amino acid sequences to those predicted to be specified by 4 of the ORFs identified in the ϕEf11 genome. These included: ORF8 (predicted scaffold protein), ORF10 (predicted major head protein), ORF15 (predicted major tail protein), and ORF23 (presumptive antireceptor).

Production of human type II collagen using an efficient baculovirus-silkworm multigene expression system.

Human type II collagen is a macromolecular protein found throughout the human body. The baculovirus expression vector system is one of the most ideal systems for the routine production and display of recombinant eukaryotic proteins in insect, larvae, and mammalian cells. We use this system to express a full-length gene, human type II collagen cDNA (4257bp), in cultured Spodoptera frugiperda 9 cells (Sf9), Bombyx mori cells, and silkworm larvae. In this study, the expression of human type II collagen gene in both insect cells and silkworm larvae was purified by nickel column chromatography, leading to 300-kDa bands in SDS-PAGE and western blotting indicative of collagen α-chains organized in a triple-helical structure. About 1mg/larva human type II collagen is purified from silkworm skin, which shows a putative large scale of collagen production way. An activity assay of recombinant human type II collagen expressed by silkworm larvae demonstrated that the recombinant protein has considerable bioactive properties. Scanning electron microscopy of purified proteins clearly reveals randomly distributed and pitted structures. We conclude that the baculovirus-silkworm multigene expression system can be used as an efficient platform for express active human type II collagen and other complicated eukaryotic proteins.

Protein Quantity and Quality of Safflower Seed Improved by NP Fertilizer and Rhizobacteria (Azospirillum and Azotobacter spp.).

HIGHLIGHTS Rhizobacteria (Azotobacter spp.) have improved the quality and quantity of safflower seed protein.Protein quality was confirmed by SDS-PAGE and new bands were found in response to different combinations of rhizobacteria and lower doses of fertilizers.The PGPR application has reduced the use of fertilizers upto 50%.Protein is an essential part of the human diet. The aim of this present study was to improve the protein quality of safflower seed by the application of plant growth promoting rhizobacteria (PGPR) in combination with conventional nitrogen and phosphate (NP) fertilizers. The seeds of two safflower cultivars Thori and Saif-32, were inoculated with Azospirillum and Azotobacter and grown under field conditions. Protein content and quality was assessed by crude protein, amino acid analysis, and SDS-PAGE. Seed crude protein and amino acids (methionine, phenylalanine, and glutamic acid) showed significant improvements (55–1250%) by Azotobacter supplemented with a quarter dose of fertilizers (BTQ) at P ≤ 0.05. Additional protein bands were induced in Thori and Saif-32 by BTQ and BTH (Azotobacter supplemented with a half dose of fertilizer) respectively. The Azospirillum in combination with half dose of fertilizer (SPH) and BTQ enhanced both indole acetic acid (IAA) (90%) and gibberellic acid (GA) (23–27%) content in safflower leaf. Taken together, these data suggest that Azospirillum and Azotobacter along with significantly reduced (up to 75%) use of NP fertilizers could improve the quality and quantity of safflower seed protein.

Analysis of Protein Sumoylation.

Sumoylation, wherein small ubiquitin-like modifier (SUMO) proteins are covalently attached to specific lysine residues of target proteins, plays an important role in regulating many diverse cellular processes via its control of the functional properties of the modified proteins. Identification of new sumoylated proteins is expected to expand understanding of the role this modification has in cell function. This unit describes two different assays for determining whether a particular protein is sumoylated: the first method employs immunoprecipitation of the protein followed by SUMO immunoblot. The second involves incubating the protein (either an in vitro translation product or a purified recombinant protein) with a reconstituted in vitro sumoylation reaction followed by examination for increased molecular-weight bands in SDS-PAGE as sumoylated forms of the protein. Either of these approaches can also be used to determine the sumoylated lysine residue(s) by comparing modification of the normal protein versus lysine-to-arginine substitutions of potential sumoylation sites, which once determined allows analysis of the effect of sumoylation on the protein's function.

A Glutathione S-transferase Elutes with Glyoxalase-I (Gly-I) During Purification of Gly-I from Maize ( Zea mays L.) Seedlings

In this study an attempt was taken to purify Glyoxalase-I (Gly-I: E.C., 4.4.1.5), from maize seedlings. Both green and non-green parts of 7 day old maize seedlings were used as plant materials. Crude proteins were precipitated by 65% (NH4)2SO4, and dialyzed overnight. The dialyzate was applied on DEAE-cellulose chromatography and eluted with linear gradient of KCl from 0 to 0.2 M. In both cases, Gly-I eluted at approximately 85 mM of KCL. The active Gly-I fractions were pooled and applied on a hydroxylapatite chromatography and eluted with 0-40 mM potassium-phosphate buffer, but the eluted fractions showed very poor activity. Therefore, the active pooled fraction of DEAE-chromatography was then applied directly on affinity chromatography (S-hexyl glutathione-agarose) for final purification and eluted with 1.2 mM of S-hexyl glutathione. The purified protein from green and non-green part had specific activity of 33.23 and 39.25 μmol min-1 mg-1 protein, respectively, along with recovery of 1.47 and 162, respectively, and yield of 83.11 and 68.15, respectively. In SDS-PAGE, the active purified affinity fraction was found to move with another protein. The spectrophotometric analysis of high active Gly-I fractions from DEAE-cellulose and affinity chromatography showed GST [another detoxifying enzyme (E.C., 2.5.1.18)] activity. This result suggested that one of the adjacent protein bands in SDS-PAGE was due to presence of a GST in Gly-I fraction.

Quantitative measurement of in vivo GSK3ß activity using phase-tag technique

Event Abstract Back to Event Quantitative measurement of in vivo GSK3ß activity using phase-tag technique Ambika Krishnankutty1*, Taeko Kimura1, Taro Saito1, Akiko Asada1, Koichi Ishiguro2 and Shin-Ichi Hisanaga1* 1 Tokyo Metropolitan University, Molecular Neuroscience, Japan 2 Juntendo University, Biology, Japan Tauopathy neurodegeneration including Alzheimer’s disease is characterized by the intracellular aggregation of hyperphosphorylated tau. Major phosphorylation sites are Ser-Pro or Thr-Pro sequences, which are catalyzed by GSK3ß and CDK5, GSK3ß generates pathological phosphorylation of tau, which is primed by physiological phosphorylation with Cdk5. Therefore, it is important to know how pathological phosphorylation by GSK3ß is induced. However, it is not fully understood how GSK3ß activity is regulated in neurons. Most previous reports estimated the GSK3ß activity using phospho-specific antibody to Ser9 inhibitory phosphorylation site. The use of the phospho-Ser9 antibody enables us to measure relative changes of the GSK3ß activity, but does not provide the absolute kinase activity. In this study, we measured active GSK3ß in various cultured cells, neurons and mouse brain using the Phos-tag technique. GSK3ß expressed in CHO-K1 cells were separated into three bands in Phos-tag SDS-PAGE; non-phosphorylated, Tyr216-phosphorylated, and Tyr216/Ser9 double-phosphorylated GSK3ß. More than 50% of GSK3ß was active with Tyr216 phosphorylation. Lithium inhibited GSK3ß by increasing Ser9 phosphorylation but a specific inhibitor SB415216 decreased active GSK3ß by inhibitory autophosphorylation at Tyr216. Insulin treatment increased Ser9 inhibitory phosphorylation but most part of GSK3ß still remained as the active form in many cultured cells including neurons. Interestingly, Ser9 phosphorylation of GSK3ß in mouse brain was different depending on brain regions, sex and age. Ser9 phosphorylation was low in hippocampus of adult female mouse. These results indicate that phospho-Ser9 immunoblotting does not measure the GSK3ß activity correctively and phospho-Tyr216 measurement by Phos-tag is instead recommended. Keywords: Hippocampus, Phosphorylation, neurodegeneration, in vivo, Phos-tag Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Krishnankutty A, Kimura T, Saito T, Asada A, Ishiguro K and Hisanaga S (2016). Quantitative measurement of in vivo GSK3ß activity using phase-tag technique. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00202 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Ms. Ambika Krishnankutty, Tokyo Metropolitan University, Molecular Neuroscience, Hachioji, Tokyo, Japan, krishnankutty-ambika@ed.tmu.ac.jp Prof. Shin-Ichi Hisanaga, Tokyo Metropolitan University, Molecular Neuroscience, Hachioji, Tokyo, Japan, Shin-ichi.Hisanaga@frontiersin.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ambika Krishnankutty Taeko Kimura Taro Saito Akiko Asada Koichi Ishiguro Shin-Ichi Hisanaga Google Ambika Krishnankutty Taeko Kimura Taro Saito Akiko Asada Koichi Ishiguro Shin-Ichi Hisanaga Google Scholar Ambika Krishnankutty Taeko Kimura Taro Saito Akiko Asada Koichi Ishiguro Shin-Ichi Hisanaga PubMed Ambika Krishnankutty Taeko Kimura Taro Saito Akiko Asada Koichi Ishiguro Shin-Ichi Hisanaga Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Expression, purification and characterization of Plasmodium falciparum vacuolar protein sorting 29

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.

Expression, purification and guanine nucleotide binding characterization of Arabidopsis RabE1d13-185 GTPase

Arabidopsis RabE1d subclass plays important plant-specific functions in plant growth and development, response to ethylene and defence to plant pathogen, besides their basic cellular role in membrane trafficking. In this study, we present the expression, purification, and characterization of the recombinant core domain of AtRabE1d13-185. AtRabE1d13-185 was successfully expressed in Escherichia coli and purified via two-step nickel affinity chromatography followed by gel filtration, and identified single band in SDS-PAGE. The resultant protein was functionally active, as determined by interaction with guanine nucleotide by a fluorescence-based assay. The intrinsic tryptophan of AtRabE1d13-185 showed fluorescence resonance energy transfer (FRET) effect upon forming complex with fluorescent methylanthraniloyl (mant)-GDP, but quenched when binding with non-labelled guanine nucleotide. The association rate of mantGDP with AtRabE1d13-185 was determined to be 3.48 × 107 s−1 M−1. The dissociation rates of GDP and mantGDP from the complex with AtRabE1d13-185 were similar. The koff values were determined to be 4.02 × 10−4 s−1 based on the FRET effect for the AtRabE1d13-185:GDP and 5.41 × 10−4 s−1 for mantGDP excited directly.

Efficient purification of lysozyme from egg white by 2-mercapto-5-benzimidazolesulfonic acid modified Fe3O4/Au nanoparticles

2-Mercapto-5-benzimidazolesulfonic acid (MBISA) modified Fe3O4/Au nanoparticles were synthesized in aqueous solution and characterized by photo correlation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The so-obtained Fe3O4/Au-MBISA nanoparticles were capable of specific adsorbing lysozyme. The maximum amount of lysozyme adsorbed on 1.0mg Fe3O4/Au-MBISA nanoparticles was 346μg. The lysozyme desorption behavior was studied and the lysozyme recovery from Fe3O4/Au-MBISA nanoparticles approached 100% under optimal conditions, and the reusability studies showed that the nanoparticles could maintain about 91% of the initial lysozyme adsorption capacity after 7 repeated adsorption–elution cycles. The Fe3O4/Au-MBISA nanoparticles were used in the purification of lysozyme from chicken egg white, which was verified by a single SDS-PAGE band. Therefore, the obtained Fe3O4/Au-MBISA nanoparticles exhibited excellent performance in the direct purification of lysozyme from egg white.

Lectin from green speckled lentil seeds (Lens culinaris) triggered apoptosis in nasopharyngeal carcinoma cell lines.

BackgroundThe green speckled lentil seed (Lens culinaris) lectin (GSLL) exhibits hemagglutinating activity, and possesses some properties distinct from those of other lentil lectins (e.g., molecular size, biological activities) that deserve further investigation. This study aims to investigate the basic properties (e.g., molecular size, amino acid sequence, sugar specificity) and biological activities (e.g., antiproliferative activity) of GSLL.MethodsGSLL was purified by successive fractionation on SP-Sepharose, Affi-gel blue gel, Mono Q, and Superdex 75. The biochemical properties of GSLL were investigated by SDS-PAGE, mass spectrometry, N-terminal amino acid sequencing, and sugar inhibition tests. For the biological activities, purified lyophilized GSLL was sterilized, adjusted to concentrations from 1 to 0 mg/mL (by twofold serial dilution) in Dulbecco’s modified Eagle’s medium with fetal bovine serum, and examined by using the MTT assay, flow cytometry, and western blotting after treatment of nasopharyngeal carcinoma CNE1 and CNE2 cell lines with the lectin.ResultsGSLL appeared as a 21-kDa band in non-reducing SDS-PAGE. It was composed of two subunits with molecular sizes of 17 and ~4 kDa. It exhibited specificity in binding to glucose and mannose, as well as glucosides and mannosides. Mass spectrometry and N-terminal amino acid sequencing revealed similarity of GSLL to L. culinaris lectin (LcL), especially higher coverage of the β-chain of LcL. A 48-h treatment with GSLL exerted antiproliferative effects on nasopharyngeal carcinoma CNE1 and CNE2 cell lines with significant inhibition at 0.125 mg/mL (P < 0.001) and 1 mg/mL (P = 0.004), respectively, and these effects were attenuated in the presence of glucose and mannose. GSLL induced apoptosis in nasopharyngeal carcinoma CNE1 cells, with detectable phosphatidylserine externalization, mitochondrial depolarization, and cell cycle arrest. Western blot analysis suggested that GSLL triggered the extrinsic apoptotic pathway involving caspase 3, 8, and 9 in CNE1 cells.ConclusionGSLL possessed some different properties from LcL (e.g., lower pI), and increased caspase 3, 8, and 9 activity in CNE1 cells.

Identification of hyaluronidase and phospholipase B in Lachesis muta rhombeata venom

Hyaluronidases contribute to local and systemic damages after envenoming, since they act as spreading factors cleaving the hyaluronan presents in the connective tissues of the victim, facilitating the diffusion of venom components. Although hyaluronidases are ubiquitous in snake venoms, they still have not been detected in transcriptomic analysis of the Lachesis venom gland and neither in the proteome of its venom performed previously. This work purified a hyaluronidase from Lachesis muta rhombeata venom whose molecular mass was estimated by SDS-PAGE to be 60 kDa. The hyaluronidase was more active at pH 6 and 37 °C when salt concentration was kept constant and more active in the presence of 0.15 M monovalent ions when the pH was kept at 6. Venom was fractionated by reversed-phase liquid chromatography (RPLC). Edman sequencing after RPLC failed to detect hyaluronidase, but identified a new serine proteinase isoform. The hyaluronidase was identified by mass spectrometry analysis of the protein bands in SDS-PAGE. Additionally, phospholipase B was identified for the first time in Lachesis genus venom. The discovery of new bioactive molecules might contribute to the design of novel drugs and biotechnology products as well as to development of more effective treatments against the envenoming.

Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus.

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 isolated from Indonesian beef. This research aimed to identify the genes involved in plantaricin IIA-1A5 production and examine its mode of action against Staphylococcus aureus. It has been reported that a bacteriocin structural gene, plnW, is present in genome of L. plantarum IIA-1A5. Here, we reported the presence of additional genes responsible for plantaricin precursor (plnA and plnEF) and a gene encoding the quorum sensor of histidine kinase (plnB). It indicates that genes involved in production of plantaricin IIA-1A5 are organized in at least two bacteriocin operons (plnABCD, plnEFI) and a structural plnW gene. Purified plantaricin IIA-1A5 yielded a single band in SDS-PAGE with apparent size of 6.4 kDa. Amino acid composition of purified plantaricin IIA-1A5 was mainly composed of cationic glutamic acid and cysteine that allowed the formation of disulphide bonds, suggesting plantaricin IIA-1A5 belongs to the pediocin-subclass of class II bacteriocins. Plantaricin IIA-1A5 displayed remarkable antibacterial activity against S. aureus, which was initiated by the adsorption of plantaricin IIA-1A5 onto the cell membrane of S. aureus. The adsorption is hypothesised to be facilitated by non-ionic interactions as it is reduced by the presence of organic solvents or detergents. This adsorption promoted leakage of cellular metabolites through the cell membrane of S. aureus, as indicated by the release of genetic and proteinaceous material of S. aureus observed at 260 and 280 nm, respectively. The leakage also promoted the release of divalent (Ca(2+), Mg(2+)) and monovalent (K(+)) cations. The release of these intracellular components might be due to pores formed in the cell membrane of S. aureus by plantaricin IIA-1A5 as shown by scanning electron microscopy. Altogether, the mode of action of plantaricin IIA-1A5 against S. aureus seems to be bactericidal as indicated by lysis of the cell membrane.

AcpM, the meromycolate extension acyl carrier protein of Mycobacterium tuberculosis, is activated by the 4'-phosphopantetheinyl transferase PptT, a potential target of the multistep mycolic acid biosynthesis.

Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4'-phosphopantetheine (4'-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4'-PP is mediated by 4' phosphopantetheinyl transfersases (PPTases). Mycobacterium tuberculosis (Mtb) possesses two essential PPTases: acyl carrier protein synthase (Mtb AcpS), which activates the multidomain fatty acid synthase I (FAS I), and Mtb PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two Mtb PPTases, AcpS or PptT, activates the meromycolate extension ACP, Mtb AcpM, en route to the production of mycolic acids, the main components of the mycobacterial cell wall. In this study, we tested the enzymatic activation of a highly purified Mtb apo-AcpM to Mtb holo-AcpM by either Mtb PptT or Mtb AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that Mtb PptT is the PPTase that activates Mtb AcpM. We measured the catalytic activity of Mtb PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by Mtb PptT was inhibited by Mtb apo-AcpM through competition for CoA, in accord with Mtb AcpM activation. A structural model of the putative interaction between Mtb PptT and Mtb AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of Mtb AcpM by Mtb PptT reveals a potential target of the multistep mycolic acid biosynthesis.

올리고당의 이해

This study was to investigate whether the bioactivity of L. plantarum L67 can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein or compounds of similar molecular weights (24 kDa). When L. plantarum L67 was treated with ZPDC glycoprotein of various concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23-kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The activities of antioxidant enzymes (SOD, GPx, and CAT), growth rate, and β-galactosidase activity were more increased at treatment with ZPDC glycoprotein than at treatment of L. plantarum L67 only, whereas the ATPase activity was decreased. Interestingly, it did not affect scavenging activity for the DPPH. The founding result suggest that, one of beneficial strategies for increase the bioactivity of L. plantarum L67 might be to take ZPDC.