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N-terminal Amino Acid Sequence (n-terminal + amino_acid_sequence)
Selected AbstractsPossible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp.FEBS JOURNAL, Issue 7 2004SIB1 in cold-adaptation A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 °C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 °C compared to that at 20 °C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N -succinyl-Ala-Leu-Pro-Phe- p -nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 °C with a kcat/Km value of 0.87 µm,1·s,1. When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T1 refolding assay at 10 and 20 °C, the protein exhibited higher activity at 10 °C with a kcat/Km value of 0.50 µm,1·s,1. These kcat/Km values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria. [source] Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp.FEMS MICROBIOLOGY LETTERS, Issue 1 2009CsO- Abstract An actinomycete strain, Amycolatopsis sp. CsO-2, produces a 27-kDa chitosanase. To reveal the molecular characteristics of the enzyme, its corresponding gene ctoA was cloned by a reverse genetic technique, based on the N-terminal amino acid sequence of the protein. The encoded CtoA protein was deduced to be composed of 286 amino acids, including a putative signal peptide (1,48), and exhibited 83% identity in the amino acid sequence with the family 46 chitosanases from Streptomyces sp. N174 or Nocardioides sp. N106. The active recombinant CtoA protein was successfully overproduced in Escherichia coli. The mutant protein E22Q, in which the glutamic acid residue 22 was replaced with glutamine, abolished the chitosanase activity, showing that the Glu22 residue is required for the enzymatic activity. CtoA exhibited antifungal activity against Rhizopus oryzae, which is known to produce chitosan probably as a cell wall component. In contrast, E22Q did not inhibit the growth of the fungus, suggesting that chitosan-hydrolyzing activity is essential for the antifungal activity. It is noteworthy that the antifungal effect of CtoA against R. oryzae was drastically enhanced by the simultaneous addition of the family 19 chitinase ChiC from Streptomyces griseus. [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] The roles of the N-terminal portions of various tachykinins in promoting salivationORAL DISEASES, Issue 4 2001K Higa OBJECTIVES: In order to determine the active sites for salivation of various tachykinins, the regulatory roles of the N-terminal portion of various newly-synthesized tachykinins were studied after i.p. injection of rats using the submandibular glands as model organs. METHODS: N-shortened oligopeptides from kassinin, eledoisin, neurokinins A (NKA) and NKB were synthesized by the multipin peptide synthesis method. Amino acids were eliminated one by one to form octa- to undeca-peptides adjoining the inactive or less active heptapeptides and various heptapeptides, in which an amino acid in position 8 (Xaa8 ), numbering as in an undecapeptide, was replaced with Tyr, Phe, Ile or Val. RESULTS: The N-terminal amino acids in positions 1 to 4 could be activators or inhibitors, depending on whether the C-terminal heptapeptide was inactive or less active. The Xaa8 residue, in combination with amino acids in positions 5 and 6, seemed to be very important in determining the sialogogic activity of a heptapeptide. The discrimination between NKA and NKB appeared due to the N-terminal amino acid sequence in positions 1 to 4 including Phe or Ser in position 6. CONCLUSIONS: It is concluded that the N-terminal amino acids in positions 1 to 4 serve as either activators or inhibitors depending upon the sialogogic activity of the C-terminal heptapeptide, in which particular amino acids in positions 5, 6 and 8 regulate its activity. [source] Barley polyamine oxidase: characterisation and analysis of the cofactor and the N-terminal amino acid sequencePHYTOCHEMICAL ANALYSIS, Issue 3 2001Anna Radová Abstract This paper reports the first purification method developed for the isolation of an homogeneous polyamine oxidase (PAO) from etiolated barley seedlings. The crude enzyme preparation was obtained after initial precipitation of the extract with protamine sulphate and ammonium sulphate. The enzyme was further purified to a final homogeneity (by the criteria of isoelectric focusing and SDS,PAGE) using techniques of low pressure chromatography followed by two FPLC steps. The purified yellow enzyme showed visible absorption maxima of a flavoprotein at 380 and 450,nm: the presence of FAD as the cofactor was further confirmed by measuring the fluorescence spectra. Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS,PAGE was 56,kDa, whilst gel permeation chromatography revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley PAO shows a high degree of similarity to that of maize PAO and to several other flavoprotein oxidases. The polyamines spermine and spermidine were the only two substrates of the enzyme with Km values 4,×,10,5 and 3,×,10,5,M and pH optima of 5.0 and 6.0, respectively. Barley polyamine oxidase is markedly inhibited by acridine dyes and hydrazines. Weak inhibition was observed with substrate analogues, aminoaldehydes, metal chelating agents and several other compounds. Copyright © 2001 John Wiley & Sons, Ltd. [source] Cloning and expression of the gene for an insect haemocyte anti-aggregation protein (VPr3), from the venom of the endoparasitic wasp, Pimpla hypochondriaca,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2009M. Paulina Dani Abstract A venom protein from the endoparasitic wasp, Pimpla hypochondriaca, was recently biochemically isolated. This protein possessed haemocyte anti-aggregation activity in vitro and shares the same N-terminal amino acid sequence as that deduced from a gene termed vpr3. The vpr3 gene was identified by sequence analysis of randomly isolated cDNAs from a P. hypochondriaca venom gland library. Presently, the gene for the full-length sequence of mature VPr3 protein was amplified from the P. hypochondriaca venom gland cDNA library by PCR. The amplicon was directionally cloned into a pET expression vector so that recombinant VPr3 (rVPr3) would have an N-terminal polyhistidine (His) tag. High levels of target protein expression were obtained following addition of IPTG (1,mM) and growth of the bacteria at 37°C for 5,h, or at 24°C for 20,h. Following lysis of bacteria grown at 37°C, the target protein partitioned into the insoluble fraction. However, at 24°C, a small amount of soluble protein was consistently detected. The amount of soluble rVPr3 was subsequently increased when the transformed bacteria were grown in Overnight Express Instant TB medium at 24°C. Soluble rVPr3 was purified utilizing the MagneHis Protein Purification System. Recombinant VPr3 was determined to have adverse effects on the cytoskeleton of Lacanobia oleracea haemocytes and to inhibit the ability of these cells to form aggregates in vitro. © 2009 Wiley Periodicals, Inc. [source] BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cellsCELLULAR MICROBIOLOGY, Issue 12 2003Asaomi Kuwae Summary The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild-type strain. The results showed that the wild-type strain, but not the type III mutant, secreted a 40-kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N-terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild-type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild-type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild-type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells. [source] Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus speciesCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000W.-L. Lin Background Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. Objective The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. Methods BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Results Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Conclusion Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts. [source] Identification and characterization of a group 2 conifer pollen allergen from Chamaecyparis obtusa, a homologue of Cry j 2 from Cryptomeria japonicaCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2000Yasueda Background Not only Cryptomeria japonica (Japanese cedar) pollen but also that of Chamaecyparis obtusa (Japanese cypress) induces the allergic symptoms of Japanese cedar pollinosis. However, allergens from C. obtusa pollen have not been as well characterized as those from C. japonica pollen. Objective We sought to identify and characterize a homologue of the second major allergen of C. japonica pollen, Cry j 2, from the pollen of C. obtusa. Methods An allergen homologous to Cry j 2 was identified in C. obtusa pollen extract by immunoblot analysis, probed with anti-Cry j 2 monoclonal antibodies and purified by a series of column chromatographic steps. Results The allergen isolated from the extract showed a slightly diffuse band of 45 kDa and closely spaced double-bands of 42 and 45 kDa on SDS-PAGE, under reducing and non-reducing conditions, respectively; the bands were approximately 5,7 kDa larger than those of Cry j 2. In 24 of 30 residues, the N-terminal amino acid sequence of the allergen was identical with corresponding sequence in Cry j 2. Most patients with pollinosis who were IgE antibody-positive to Cry j 2 were shown to be IgE antibody-positive to this allergen, and the IgE antibody levels to both allergens were highly correlated. Conclusion The results indicate that the allergen isolated from C. obtusa pollen in this study is a homologue of Cry j 2. The allergen was designated as Cha o 2 according to the WHO/IUIS Allergen Nomenclature Subcommittee recommendation. [source] Detection of tmRNA-mediated trans-translation products in Bacillus subtilisGENES TO CELLS, Issue 3 2002Ai Fujihara Background: Bacterial tmRNA (10Sa RNA) is involved in a trans -translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans -translation. Results: The wild-type tmRNA gene (ssrA) in the genome was replaced by a variant ssrA encoding a tag-peptide sequence containing six histidine residues (His-tag) and two aspartic acids at the C-terminus. The His-tagged proteins that accumulated in the cells without degradation were fractionated by Ni2+ -NTA column and gel electrophoresis and were detected by Western blotting with an anti-His-tag antibody. The results showed that the trans -translation occurred more frequently at a high temperature (50 °C) than at a low temperature (37 °C). Two-dimensional (2D) gel electrophoresis of the products revealed many distinct spots, which represent specific target proteins for the trans -translation reaction. Furthermore, the 2D gel patterns of the products from cells cultured at high and low temperatures were apparently different. Several tagged proteins were identified by the N-terminal amino acid sequences of the products. Conclusion:Trans -translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures. [source] Comparative analysis of two biliproteins, BP1 and BP2, from haemolymph of cabbage white butterfly, Pieris rapaeARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2006Chi Won Choi Abstract Two blue-pigment binding proteins, BP1 and BP2, are present in larval and pupal haemolymph of cabbage white butterfly, Pieris rapae, and fluctuate in expression during development. Both BP1 and BP2 are found in pupal haemolymph in varying proportions as well as in adult haemolymph, while only small amounts of BP2 are found in larval haemolymph. BPs are separated by 75% ammonium sulfate, and then purified effectively by ion exchange column chromatography and preparative gel electrophoresis. It was shown that BP1 and BP2 have molecular masses of 20,244 and 19,878 Da, and isoelectric points of 7.0 and 6.8, respectively. Considering their amino acid compositions and N-terminal amino acid sequences, the two proteins are almost identical except the first N-terminal amino acid. The first amino acid of BP1 is asparagine, whereas the initial residue of BP2 is aspartic acid. Anti-BP1 cross-reacts with BP2, indicating that they have immunological homogeneity. Western blotting analyses revealed that only BP1 was present in the larval tissues such as fat body, integument, muscle, and hindgut. However, BP1 was not found in midgut, Malphigian tubules, and silk gland. BP1 was also present in the protein bodies, and both cuticle and hemocoel sides of larval epidermis cells by the transmission electron microscopic observation. The information in this report will facilitate studies on the molecular biology and biological significance of insect BPs. Arch. Insect Biochem. Physiol. 61:220,230, 2006. © 2006 Wiley-Liss, Inc. [source] What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?CLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2002A. Dìaz-Perales Background Class I chitinases are the major panallergens in fruits associated with the latex,fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial. Objective We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model. Methods Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed. Results Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex,fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70,90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results. Conclusions Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens. [source] | ||||||||||||||||