Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

ABSTRACT Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed toGalleria mellonella larvae.

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Preparative isolation and characterization of the B875 complex from Rhodobacter sphaeroides 2.4.1

Biochemistry and Cell Biology ◽

10.1139/o88-053 ◽

1988 ◽

Vol 66 (5) ◽

pp. 442-448 ◽

Cited By ~ 14

Author(s):

Rafael Picorel ◽

Gabriel Gingras

Keyword(s):

Rhodobacter Sphaeroides ◽

Phosphatidyl Ethanolamine ◽

Sodium Dodecyl ◽

Pigment Analysis ◽

Preparative Isolation ◽

Ethanolamine Phosphatidyl ◽

Isolation And Characterization ◽

Detergent System ◽

Triton X

We have developed a simple and efficient method, using a mixed detergent system of sodium dodecyl sulfate and Triton X-100, for the preparative isolation of theB875 complex from Rhodobacter sphaeroides 2.4.1. As a bonus, the method allows the preparation of both the B875 and B800-850 complexes from the same batch of chromatophores. The preparations are spectrally pure, as indicated by absorption and circular dichroism spectroscopy. The latter method suggests that the Qy band of the B875 complex is due to weakly interacting bacteriochlorophyll molecules. Protein and pigment analysis shows that the B875 complex contains 2 mol of bacteriochlorophyll and 2 mol of sphaeroidene per mol of apoprotein (12 266 g), whereas the B800-850 complex contains 3 mol of bacteriochlorophyll and 1 mol of sphaeroidene per mol of apoprotein (11 497 g). While these stoichiometries are in accord with currently accepted models, they disagree with their published experimental basis. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol were found to be present in the B875 complex.

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Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes

Biochemical Journal ◽

10.1042/bj3190887 ◽

1996 ◽

Vol 319 (3) ◽

pp. 887-896 ◽

Cited By ~ 35

Author(s):

Edward T PARKIN ◽

Anthony J TURNER ◽

Nigel M HOOPER

Keyword(s):

Light Scattering ◽

Gradient Centrifugation ◽

Buoyant Density ◽

Annexin V ◽

Density Fraction ◽

Calcium Dependent ◽

Membrane Complex ◽

Isolation And Characterization ◽

Insoluble Complex ◽

Marker Proteins

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5´-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca2+-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the metal chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.

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Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes

Biochemical Journal ◽

10.1042/bj2070037 ◽

1982 ◽

Vol 207 (1) ◽

pp. 37-41 ◽

Cited By ~ 14

Author(s):

A Imam ◽

D J Laurence ◽

A M Neville

Keyword(s):

Human Milk ◽

Milk Fat ◽

Sodium Dodecyl ◽

Membrane Glycoproteins ◽

Lectin Affinity Chromatography ◽

Isolation And Characterization ◽

Milk Fat Globule ◽

Fat Globule ◽

Milk Fat Globule Membranes ◽

Human Milk Fat

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed ‘epithelial-membrane glycoprotein’ (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.

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Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

Biochemistry and Cell Biology ◽

10.1139/o86-004 ◽

1986 ◽

Vol 64 (1) ◽

pp. 21-28 ◽

Cited By ~ 85

Author(s):

Malcolm B. Perry ◽

Leann MacLean ◽

Douglas W. Griffith

Keyword(s):

Escherichia Coli ◽

Brucella Abortus ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Extraction Procedure ◽

Periodate Oxidation ◽

Cross Reactivity ◽

E Coli ◽

Structure Formula ◽

Phenol Water

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol–water extraction procedure was shown by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure:[Formula: see text]The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.

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Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

Biochemical Journal ◽

10.1042/bj2280111 ◽

1985 ◽

Vol 228 (1) ◽

pp. 111-117 ◽

Cited By ~ 10

Author(s):

E Davies Jones ◽

F A Hashim ◽

Y Kajita ◽

F M Creagh ◽

P R Buckland ◽

...

Keyword(s):

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Water Soluble ◽

Tsh Receptor ◽

Human Thyroid ◽

Thyrotropin Receptor ◽

Freezing And Thawing ◽

Sucrose Density Gradient Centrifugation ◽

A Subunit

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

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Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

Applied and Environmental Microbiology ◽

10.1128/aem.00409-09 ◽

2009 ◽

Vol 75 (14) ◽

pp. 4661-4667 ◽

Cited By ~ 26

Author(s):

Alejandro Hernández-Soto ◽

M. Cristina Del Rincón-Castro ◽

Ana M. Espinoza ◽

Jorge E. Ibarra

Keyword(s):

Bacillus Thuringiensis ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Microbial Control ◽

Control Agent ◽

Mass Spectrometry Analysis ◽

Parasporal Crystal ◽

Spectrometry Analysis ◽

Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

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A glycoprotein associated with the membrane fraction of human B but not T lymphocytes.

Journal of Experimental Medicine ◽

10.1084/jem.142.6.1416 ◽

1975 ◽

Vol 142 (6) ◽

pp. 1416-1424 ◽

Cited By ~ 6

Author(s):

S Fujita ◽

S D Litwin ◽

N Hartman

Keyword(s):

Membrane Fraction ◽

Gradient Centrifugation ◽

Human Lymphocytes ◽

Sodium Dodecyl ◽

Protein Band ◽

Sucrose Density Gradient ◽

Peripheral Blood Lymphocytes ◽

Sucrose Density Gradient Centrifugation ◽

Lymphoid Lines

A method is described which employs differential centrifugation and sucrose density gradient centrifugation to isolate a membrane fraction from human lymphocytes. Membrane preparations from long-term human cultured B- and T-lymphoid lines, peripheral blood lymphocytes, tonsillar lymphocytes, and thymocytes were analyzed on 0.5% sodium dodecyl sulfate-7.5% polyacrylamide gels stained for protein and carbohydrate. The most important finding was a major glycoprotein of approximately 30,000 daltons associated with the membrane preparations from B lymphocytes. T-lymphocyte preparations did not contain readily detectable amounts of this membrane-associated component. The T-cell lymphoid line MOLT-4 was unique in that it had a narrow protein band at approximately 30,000 daltons which did not contain carbohydrate.

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Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

Infection and Immunity ◽

10.1128/iai.66.9.4374-4381.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4374-4381 ◽

Cited By ~ 58

Author(s):

John C. McMichael ◽

Michael J. Fiske ◽

Ross A. Fredenburg ◽

Deb N. Chakravarti ◽

Karl R. VanDerMeid ◽

...

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Bacterial Attachment ◽

Vaccine Candidates ◽

Isolation And Characterization ◽

Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

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Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

KnE Life Sciences ◽

10.18502/kls.v3i5.986 ◽

2017 ◽

Vol 3 (5) ◽

pp. 139

Author(s):

Mariana Wahjudi ◽

Catherina . ◽

Nita Marcelia Wangunhardjo ◽

Ernest Suryadjaja ◽

Xavier Daniel

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Recombinant Plasmid ◽

Sodium Dodecyl ◽

Xylanase Activity ◽

Cellular Protein ◽

Host Cells ◽

Chromosomal Dna ◽

Clear Zone ◽

Sds Page

The xynB gene of Bacillus subtilis subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the xynB gene from Bacillus subtilis subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the xynB gene in Escherichia coli Origami as host cells. The xynB gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The xynB gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for xynB gene and for the vector, then continued by restriction analyses. The result showed that transformants clone 9 and 10 bear the recombinant pMMB-xynB plasmid. The xylanase activity of xynB gene in Escherichia coli Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-xynB recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the xynB gene of Bacillus subtilis subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the Escherichia coli Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.

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Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

Indian Journal of Fisheries ◽

10.21077/ijf.2019.67.2.84594-08 ◽

2020 ◽

Vol 67 (2) ◽

Author(s):

Snatashree Mohanty ◽

M. Makesh ◽

K. V. Rajendran ◽

P. P. Suresh Babu ◽

Deepika Anand ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Reactivity ◽

Catla Catla ◽

Cirrhinus Mrigala ◽

Indian Major Carps ◽

Sds Page ◽

Igg1 Isotype ◽

Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

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