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Filtration Chromatography (filtration + chromatography)
Kinds of Filtration Chromatography Selected AbstractsSeparation of Pure and Immunoreactive Virus-Like Particles Using Gel Filtration Chromatography Following Immobilized Metal Ion Affinity ChromatographyBIOTECHNOLOGY PROGRESS, Issue 2 2001Yu-Shen Cheng A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni2+) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm3. However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus. [source] Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cellsCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2000Jacquet Background The major house dust mite allergen Der p 1 elicits strong IgE antibody responses in patients suffering from mite allergy. Objective This study reports the expression and characterization of a recombinant precursor form of Der p 1 secreted as ProDer p 1 from insect cells. Methods The cDNA coding for ProDer p 1 was cloned downstream to the gp67 signal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplified ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mite culture, in terms of glycosylation, enzymatic activity as well as IgG- and IgE-binding capacity. Results Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns were also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydrate analysis clearly indicated that ProDer p 1 expressed from insect cells was glycosylated and that glycan structures were located only in the prosequence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclonal and polyclonal IgG antibodies compared to natural Der p 1. Specific activity measurements using synthetic substrates clearly indicated that, contrary to natural Der p 1, ProDer p 1 was totally enzymatically inactive. Conclusions The expression of an enzymatically inactive and highly antigenic ProDer p 1 zymogen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy. [source] Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cellsFEBS JOURNAL, Issue 1 2006Ryo Yokoyama Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is ,,680 000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits. [source] Biochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvaceaFEBS JOURNAL, Issue 2 2004Shicheng Chen We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ,,20,30% of peak levels throughout the subsequent stages of sporophore development. [source] The histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pHFEBS JOURNAL, Issue 10 2003Gregorio Fernández-Ballester The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ,G = 4.0 ± 0.2 kcal·mol,1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7,9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5,4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family. [source] Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoidesFEBS JOURNAL, Issue 22 2002Nicolas Sauvageot The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a ,2,2,2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 µm. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway. [source] Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalisFEBS JOURNAL, Issue 22 2001Mari Yotsu-Yamashita Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 ± 0.97 nmol·mg,1 protein and a Kd of 14.6 ± 0.33 nm for [3H]saxitoxin in equilibrium binding assays. [3H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 µm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish. [source] Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycleFEBS JOURNAL, Issue 17 2001Shin-ichi Yokota The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of ,, , and ,-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the , and , subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower , and , subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the , and , subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of , and , subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells. [source] Purification and characterization of a glutathione S -transferase from the fungus Cunninghamella elegansFEMS MICROBIOLOGY LETTERS, Issue 2 2001Chang-Jun Cha Abstract Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S -transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120,000×g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate,polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of Mr 27,000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian ,-, ,-, and ,-class GSTs, although it showed a small degree of cross-reactivity with a ,-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs. [source] Inhibition of endive (Cichorium endivia L.) polyphenoloxidase by a Carica papaya latex preparationINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2001David De Rigal When endive polyphenoloxidase (PPO) was incubated with a crude papaya latex extract, it rapidly lost its activity. Inactivation was ascribed to thermostable nonenzymatic factors of low molecular weight. These factors were partially purified by a two step protocol including gel filtration chromatography on Biogel P2 and ion exchange chromatography using DEAE Sephadex A25. The PPO-inactivation rate was first order, when either inactivating agent or proton concentration was evaluated. Inactivation could be partially reversed by CuSO4, which suggested that the inactivating factor(s) bound to the copper site of the enzyme. On a more rapid time scale than inactivation, papaya latex extract acted also as a weak noncompetitive PPO inhibitor. [source] Measurement-specific bioavailable testosterone using concanavalin A precipitation: Comparison of calculated and assayed bioavailable testosteroneINTERNATIONAL JOURNAL OF UROLOGY, Issue 11 2009Kenrou Yamamoto Objective: To assess the value of calculated bioavailable testosterone (cBT) and assayed BT (aBT) for the diagnosis of late-onset hypogonadism (LOH) in middle-aged and elderly subjects. Methods: In order to assay serum BT, sex hormone-binding globulin was precipitated with concanavalin-A and then testosterone was measured using liquid chromatography-tandem mass spectrometry. To validate the non-sex-hormone-binding-globulin-bound testosterone, gel filtration chromatography and concanavalin-A sepharose were used. Following this validation, the usefulness between aBT and cBT was evaluated in clinical samples. Results: Eighty-eight healthy male volunteers (mean age 65.6 years, range: 50,86) were recruited for this study. A significant correlation was found between cBT and aBT (R2 = 0.53, P < 0.01). Mean value ratio (cBT/aBT) was 2.48. Both cBT (R2 = 0.122) and aBT (R2 = 0.251) decreased with age. Variations in aBT were less marked than those for cBT, suggesting that aBT can be used to determine age-related reduced testosterone levels. Conclusion: aBT levels are more reliable than cBT levels for the diagnosis of LOH in middle-aged and elderly subjects. [source] Isolation and characterization of a protease from Pseudomonas fluorescens RO98JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2000R. Koka Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 °C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 °C, and pH 4·5 and 9·0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5·0 and 35 °C, respectively. It was heat stable with a D55 of 41 min and a D62·5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144·28, 18·73, 110·20 and 35·23 µmol were obtained for whole, ,-, ,- and ,-casein, with a Vmax of 8·26, 0·09, 0·42 and 0·70 µmol mg,1 min,1, respectively. The enzyme hydrolysed ,-casein preferentially when incubated with artificial casein micelles. [source] Novel thermostable serine collagenase from Thermoactinomyces sp.JOURNAL OF BASIC MICROBIOLOGY, Issue 4 200621E: purification, some properties A thermophilic actinomycete strain Thermoactinomyces sp. 21E producing a highly thermostable serine collagenase was isolated from Bulgarian soil. The collagenase, produced extracellular by Thermoactinomyces sp. 21E, was purified to homogeneity by heat treatment, ultrafiltration, saturation with ammonium sulfate and gel filtration chromatography with a 101-fold increase in specific activity and 58% recovery. The collagenase has a relative molecular mass of 50000 by SDS-PAGE. The optimum temperature for the enzyme activity was 60,65 °C in the absence of Ca2+ and 70,75 °C in the presence of Ca2+. About 40% of the original activity remaining after incubation at 85 °C for 30 min in the presence of Ca2+. The optimum pH for the enzyme activity was 9.0,9.5 and the enzyme was stable for 1h at 70 °C in the pH range from 7.5 to 12.5. The collagenase was strongly inhibited by active-site inhibitors of serine protease PMSF and DFP, which indicated that the enzyme is serine protease. The enzyme activity was completely inhibited by Hg2+, Cu2+ and Fe2+. However, Ca2+ strongly activated the collagenase activity. The collagenase from Thermoactinomyces sp. 21E showed high activity toward type I collagen, acid-soluble collagen, gelatin and Pz-PLGPR. However, elastin for collagenase was inert as substrate. The properties of the collagenase from strain 21E suggest that this enzyme is a new collagenolytic protease that differs from the collagenases and serine proteases reported so far. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Purification and characterization of recombinant human erythropoietin from milk of transgenic pigsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2009Eun Gyo Lee Abstract BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal-derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from the milk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse-phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and > 98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms and was underglycosylated in contrast to CHO-derived rhEPO. Cell proliferation of the F-36/EPO-dependent cell line was proportional to the dose of transgenic pig-derived rhEPO. CONCLUSION: Transgenic pig-derived rhEPO with high purity was achieved after three-step chromatography following two-step precipitation. The transgenic pig-derived rhEPO was demonstrated to have comparable potency with CHO-derived rhEPO. Transgenic pig-derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo. Copyright © 2008 Society of Chemical Industry [source] Effect of substrate size on immunoinhibition of amylase activity,JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 2 2001Ilka Warshawsky Abstract Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10‐fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one. J. Clin. Lab. Anal. 15:64–70, 2001. [source] PURIFICATION AND CHARACTERIZATION OF ,-CARRAGEENASE FROM MARINE BACTERIUM MUTANT STRAIN PSEUDOALTEROMONAS SP.JOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2010AJ5-13 AND ITS DEGRADED PRODUCTS ABSTRACT A ,-carrageenan-degrading bacterial strain AJ5 isolated from the intestine of Apostichopus japonicus was identified as Pseudoalteromonas sp. based on the phenotypic characters and 16S rRNA gene sequencing. The mutant Pseudoalteromonas sp. AJ5-13 with ,-carrageenase activity of 61 U/mg protein was obtained from Pseudoalteromonas sp. AJ5 using mutagenesis technique. An extracellular ,-carrageenase was purified from Pseudoalteromonas sp. AJ5-13 cultural supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-200) and cation-exchange chromatography (CM-cellulose 52). The purified enzyme yielded a single band on SDS-PAGE with the molecular mass of 35 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel ,-carrageenase. The pI and Km of the enzyme were 8.5 and 9.8 ± 0.2 mg/mL, respectively. The enzyme exhibited maximal activity at pH 8.0 and 55C. It hydrolyzed the ,-1, 4-glycosidic linkages of ,-carrageenan yielding ,-neocarrabiose, -tetraose, -hexaose, -octaose and -decaose sulfates as the main end-products. PRACTICAL APPLICATIONS ,-Carrageenases degrade ,-carrageenan by hydrolyzing the ,-1,4 linkages to a series of oligosaccharides. Thus, it is expected that like other ,-carrageenases, the ,-carrageenase isolated from Pseudoalteromonas sp. AJ5-13 would also be useful in seaweed biotechnology, pharmacy and immunology. ,-Carrageenases can be applied to study the composition and structure of carrageenans from different red alga, and to study the bacterial ,-carrageenan metabolism. They also provide the opportunity to investigate the structure-function relationship of the hydrolases that degrade self-associating sulfated polysaccharides. Examples of the practical applications of ,-carrageenases include their use in degrading the cell walls of seaweeds to obtain protoplasts, and in hydrolyzing ,-carrageenan to produce oligosaccharides. ,-Carrageenan-oligosaccharides have various potential biological properties, such as antiviral, antitumor, antioxidant activities, cytoprotection, immunomodulation, etc. [source] Comparative Structural, Emulsifying, and Biological Properties of 2 Major Canola Proteins, Cruciferin and NapinJOURNAL OF FOOD SCIENCE, Issue 3 2008J. Wu ABSTRACT:, Canola is an economically important farm-gate crop in Canada. To further explore the potential of canola protein as value-added food and nutraceutical ingredients, a better understanding of fundamental properties of 2 major canola proteins is necessary. Two major protein components, cruciferin and napin, were isolated from defatted canola meal by Sephacryl S-300 gel filtration chromatography. SDS-PAGE showed that cruciferin consists of more than 10 polypeptides, and noncovalent links are more important than disulphide bonds in stabilizing the structural conformation. Napin consists of 2 polypeptides and is stabilized primarily by disulphide bonds. Purified cruciferin showed 1 major endothermic peak at 91 °C compared with that of 110 °C for napin. Emulsion prepared by cruciferin showed significant higher specific surface area and lower particle size than that of napin. The study indicated that the presence of napin could detrimentally affect the emulsion stability of canola protein isolates. Hydrolysates from cruciferin and napin showed potent angiotensin I-converting enzyme inhibitory activity (IC50: 0.035 and 0.029 mg/mL, respectively), but weaker than that of canola protein isolate hydrolysate (IC50: 0.015 mg/mL). [source] Soy-Derived Immunoglobulin Production Stimulating Factor Enhances IgM Production of Mouse Spleen LymphocytesJOURNAL OF FOOD SCIENCE, Issue 7 2006N. Maeda ABSTRACT:, We have reported previously that some proteins such as lactoferrin and lysozyme control the immune system via immunoglobulin (Ig) production. In the course of the study about the function of dietary proteins and peptides, Ig production-stimulating activity of an unknown protein contained in soybean trypsin inhibitor (STI) preparation was found. Thus, we examined the activity of the unknown activator and the mechanism of the activity. The factor significantly elevated IgM production by mouse spleen lymphocytes in a dose-dependent manner. In addition, the unknown activator up-regulated the expression of interleukin (IL)-6 and IL-10 mRNA. And IgM production enhancing activity of STI was significantly suppressed by neutralization of IL-6. Then, to clarify whether STI itself is the active compound or not, STI was fractionized by gel filtration chromatography. We found that the activity the content of STI did not correlate with and the active fractions contained some proteins whose molecular weight is more than 20 kDa. These suggest that an unknown activator exists in STI preparation. [source] Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spiculesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2005S. Lilla Abstract A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined. Copyright © 2005 John Wiley & Sons, Ltd. [source] Isolation and characterization of antimicrobial proteins and peptide from chicken liverJOURNAL OF PEPTIDE SCIENCE, Issue 6 2007Guan-Hong Li Abstract Endogenous antimicrobial peptides and proteins are crucial components of the innate immune system and play an essential role in the defense against infection. Antimicrobial activity was detected in the acid extract of livers harvested from healthy adult White Leghorn hens, Gallus gallus. Two antimicrobial proteins and one antimicrobial polypeptide were isolated from the liver extract by cation-exchange and gel filtration chromatography, followed by two-step reverse-phase high-performance liquid chromatography (RP-HPLC). These antimicrobial components were identified as histones H2A and H2B.V, and histone H2B C -terminal fragment using peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry. The proteins and the peptide identified in the present study, which exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, were thermostable and showed salt-resistant activity. The antimicrobial properties of histones and histone fragment in chicken provide further evidence that histones, in addition to their role in nucleosome formation, may play an important role in innate host defense against intracellular or extracellular microbe invasion in a wide range of animal species. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Hemagglutinating activity and corresponding putative sequence identity from Curcuma aromatica rhizomeJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2008Ponpimol Tiptara Abstract BACKGROUND:Curcuma aromatica is a medicinal plant belonging to the Zingiberaceae family with an incomplete genome sequence. It has been reported that extract from the rhizome of this plant contains haemagglutinating activity. In this study the profile of fractions containing hemagglutinating activity is described. RESULTS: Following extraction with saline buffer, the protein solution was fractionated by ammonium sulfate precipitation. Ion-exchange chromatography was completed on fast-flow SP-Sepharose, as well as gel filtration chromatography on Superdex 75. The active fractions were then separated by one-dimensional sodium dodecyl sulfate,polyacrylamide gel electrophoresis and labeled proteins were digested with trypsin. The digest bands were analyzed by reversed-phase liquid chromatography,tandem mass spectrometry. Inferred peptide sequences were used in Mascot searching and mass spectrometry-driven BLAST (MS-BLAST) homology searches allowed the recognition of related proteins in other species of Viridiplantae. Six putative proteins from nine bands showed similarity with lectin sequences. CONCLUSION: This study reports the identification of six lectins from the Curcuma aromatica rhizome achieved by mass spectrometry using MS-BLAST algorithms to search for homology between de novo determined peptide sequences and protein sequences available in sequence databases. Copyright © 2008 Society of Chemical Industry [source] Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2004Augustine C Ogbonna Abstract A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion-exchange chromatography on Q- and S-Sepharose (fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 8.4-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg,1 protein. SDS,PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0,8.0 and retained over 60% activity at 70 °C after 30-min incubation. It was highly significantly (P < 0.001) inhibited by Hg2+, appreciably (P < 0.01) inhibited by Ag+, Ba2+ and Pb2+ but highly significantly (P < 0.001) activated by Co2+, Mn2+ and Sr2+. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p -chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: Km = 0.33 mg ml,1; Vmax = 0.08 µmol ml,1 min,1. Copyright © 2004 Society of Chemical Industry [source] Characterization of a bacteriocin produced by Prevotella nigrescens ATCC 25261LETTERS IN APPLIED MICROBIOLOGY, Issue 5 2004J. Kaewsrichan Abstract Aims:, To characterize the antimicrobial activity produced by Prevotella nigrescens ATCC 25261, and to evaluate its safety on cultured gingival fibroblasts. Methods and Results:, An antimicrobial activity was obtained from purifying the culture supernatant of Pr. nigrescens ATCC 25261. Purification of the active compound was achieved with ammonium sulphate precipitation followed by anion-exchange and gel filtration chromatography. As revealed by SDS-PAGE, the active fraction was relatively homogeneous, showing a protein with an approximate molecular weight of 41 kDa. The antimicrobial compound, named nigrescin, exhibited a bactericidal mode of action against Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, and Actinomyces spp. Nigrescin was stable in a pH range between 6·5 and 9·5, at 100°C for 10 min, and resistant to lyophilization. But its activity was lost after proteinase K treatment. Despite at very high concentrations beyond the minimum inhibitory concentration (MIC), nigrescin was not toxic to the gingival fibroblasts. Conclusion:, Nigrescin is a novel bacteriocin produced by Pr. nigrescens ATCC 25261. It exhibits antimicrobial activity against species that are implicated in periodontal diseases. The absence of toxicity on the gingival fibroblasts suggests the possibility in using of nigrescin for an application in periodontal treatment. Significance and Impact of the Study:, Novel evidence on nigrescin would make Pr. nigrescens ATCC 25261 attractive in biotechnological applications as an antimicrobial agent in clinical dentistry. [source] Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2004Sofía Fraile Summary The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic ,-helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope-tagged version of the 23 kDa C-terminal secretion signal of haemolysin (EHlyA). The zipper-containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross-linking experiments and coexpression with a secretion-deficient derivative of ZEHlyA indicated that leucine zipper-dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH -domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHH moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH -EHlyA and VHH -ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH -ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10-fold increase in their avidity. These data reveal a non-anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity. [source] Immediate hypersensitivity to Malassezia furfur in patients with atopic dermatitisMYCOSES, Issue 4 2007A. R. Khosravi Summary Atopic dermatitis (AD) is a chronic pruritic dermatitis that has unknown aetiology. It seems that Malassezia furfur has a role in pathogenesis of AD. The purpose of this study was to evaluate skin responses to M. furfur antigens in AD patients. Malassezia furfur was grown and the yeasts were broken. Cells were centrifuged and supernatants were used as crude extracts (CE). Protein components of CE were separated by sodium dodecylsulphate,polyacrylamide gel electrophoresis (SDS,PAGE). In addition, to fractionate CE antigens, gel filtration chromatography was performed. One hundred and fifteen AD patients were selected for skin-prick test (SPT). In SDS,PAGE, CE showed a total of 19 different protein bands (10,100 kDa). Chromatographic gel filtration with M. furfur proteins showed four major fractions (F). The protein pattern of F1 (tube no. 40) was between 22 and 100 kDa and it was selected for SPT. In SPT, 49.6% and 42.6% patients showed positive reactions with CE and F1 antigens respectively. The most positive results were obtained in 20,29 aged group (P < 0.001). The allergens of M. furfur may have a role in AD signs; it is suggested to use F1 antigens in allergy tests. [source] Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortusPARASITE IMMUNOLOGY, Issue 2 2000Smith Jacalin lectin was used as a ligand to isolate a fraction containing two distinct protective antigens from detergent extracts of membranes from Haemonchus contortus. The first antigen was identified as a complex which appeared very similar to Haemonchus galactose-containing glycoprotein (H-gal-GP), which is a previously described protective protease complex, except that it was substantially depleted of one of the main H-gal-GP components, a 230 kDa metallopeptidase-containing band. The new complex was termed Haemonchus sialylated galactose-containing glycoprotein (H-sialgal-GP), because it bound to jacalin but not to peanut lectin and only jacalin will bind the sialylated form of galactosyl (,-1,3) N -acetylgalactosamine. Two protection trials with sheep showed that H-sialgal-GP and H-gal-GP were equally efficacious, reducing numbers of Haemonchus eggs by between 86% and 93% and worms by between 52% and 75%, respectively. The second jacalin-binding protective antigen fraction was separated from H-sialgal-GP by ion exchange and gel filtration chromatography. It was greatly enriched for two proteins termed p46 and p52 according to their apparent molecular weights. Immunization of sheep with these proteins gave protection values of 78% for eggs and 33% for worms, which are significantly lower than those obtained with either H-gal-GP or H-sialgal-GP. N -terminal amino acid sequence data from p46 and p52 showed that both proteins were closely related to a previously described 45 kDa Haemonchus membrane protein, which had conferred protection against Haemonchus in guinea-pigs. [source] Purification and characterization of a subtilisin-like serine protease induced during the senescence of wheat leavesPHYSIOLOGIA PLANTARUM, Issue 4 2003Irma N. Roberts A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg,1 h,1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65,75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process. [source] Characterization of bacteriocin produced by Streptococcus bovis J2 40-2 isolated from traditional fermented milk ,Dahi'ANIMAL SCIENCE JOURNAL, Issue 1 2009Md Harun-ur RASHID ABSTRACT A bacteriocin-producing strain Streptococcus bovis J2 40-2 was isolated from traditional fermented milk ,Dahi' in Bangladesh. Despite its narrow antimicrobial spectrum, it showed strong antimicrobial activity against extremely challenging and problematic organisms in foods, such as Listeria monocytogenes. Bacteriocin was sensitive to several proteolytic enzymes and showed antimicrobial activity over a wide pH range of 2.0,10.0. It was stable when heated to 110°C for 20 min, but lost 25% of its activity when heated to 121°C for 15 or 20 min. Optimum bacteriocin production (5600 AU/mL) was achieved when the strain was cultured at 37°C for 24 h in MRS medium rather than in TYLG, GM17, or skim milk medium. Bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut-off MW: 3500) and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that bacteriocin had a molecular weight of approximately 4.5 kDa. [source] Development of a method to assess binding of astaxanthin to Atlantic salmon Salmo salar L. muscle proteinsAQUACULTURE RESEARCH, Issue 4 2005Madhury R Saha Abstract Several methods were examined to characterize the binding between astaxanthin and salmon muscle protein(s) in order to provide tools for evaluation of the role of muscle proteins on astaxanthin retention in Atlantic salmon Salmo salar L. flesh. The methods included gel filtration chromatography, displacement of a hydrophobic probe and ultrafiltration. With gel filtration chromatography, aggregation of astaxanthin under the experimental conditions was a major problem for the separation of bound astaxanthin from free astaxanthin because the apparent molecular weight of aggregated astaxanthin or astaxanthin micelles was in the range of protein,astaxanthin complexes. Displacement of the fluorescent probe 8-anilino-1-naphthalenesulphonate (ANS) was not effective as astaxanthin quenched the fluorophore so that displacement could not be observed. An ultrafiltration method was developed using 200-mM sodium cholate for dispersion of astaxanthin aggregates. This allowed unbound astaxanthin to be separated from bound astaxanthin using a 30-kDa filter. After salmon muscle proteins were solubilized in different fractions by sequential extraction using low ionic strength solutions, the astaxanthin binding of different fractions was assessed using the ultrafiltration method. The significant difference (P<0.05) observed in the astaxanthin binding of the various fractions suggests an application of this assay to detect differences in affinity of proteins for astaxanthin. The results also suggest that proteins other than actomyosin or actin can bind astaxanthin in Atlantic salmon flesh. This method can be used for the identification of astaxanthin-binding proteins in salmon flesh and other tissues. [source] Purification and properties of trypsin-like enzyme from the midgut of Morimus funereus (coleoptera, cerambycidae) LarvaeARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Nikola Lon Abstract Trypsin-like enzyme (TLE) from the anterior midgut of Morimus funereus larvae was purified by anion exchange chromatography and gel filtration chromatography and characterized. Specific TLE activity was increased 322-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 9.0 (optimum pH range 8.5,9.5) and temperature optimum of 45°C with the KM ratio of 0.065,mM for benzoyl-arginine- p -nitroanilide (BApNA). Among a number of inhibitors tested, the most efficient was benzamidine (KI value of 0.012,mM, Ic50 value of 0.204,mM) while inhibition of TLE activity by SBTI, TLCK, and PMSF was partial. Almost all divalent cations tested enhanced the enzyme activity, amongst them Co2+ and Mn2+ stimulated TLE activity for 2.5 times. The purified TLE (after gel-filtration on Superose 12 column) had a molecular mass of 37.5,kDa with an isoelectric point over 9.3. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 38,kDa, suggesting that the enzyme is a monomer. © 2010 Wiley Periodicals, Inc. [source] | |||||||||||||||||||||||||||||||