scholarly journals Bacteriocin Markers for Propionibacteria Gene Transfer Systems

2000 ◽  
Author(s):  
Susan F. Barefoot ◽  
Bonita A. Glatz ◽  
Nathan Gollop ◽  
Thomas A. Hughes
Keyword(s):  
Biological Activity ◽  
Gene Transfer ◽  
Indirect Elisa ◽  
Polyclonal Antibodies ◽  
Transmembrane Protein ◽  
Basic Knowledge ◽  
N Terminus ◽  
Charged Residues ◽  
Dairy Propionibacteria ◽  
Jenseniin G

The antibotulinal baceriocins, propionicin PLG-1 and jenseniin G., were the first to be identified, purified and characterized for the dairy propionibaceria and are produced by Propionibacterium thoenii P127 and P. thoenii/jensenii P126, respectively. Objectives of this project were to (a) produce polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1; (b) identify, clone and characterize the propionicin PLG-1 (plg-1) and jenseniin G (jnG) genes; and (3) develop gene transfer systems for dairy propionibacteria using them as models. Polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1 were produced in rabbits. Anti-PLG-1 antiserum had high titers (256,000 to 512,000), neutralized PLG-1 activity, and detected purified PLG-1 at 0.10 mg/ml (indirect ELISA) and 0.033 mg/ml (competitive indirect ELISA). Thirty-nine of 158 strains (most P. thoenii or P. jensenii) yielded cross-reacting material; four strains of P. thoenii, including two previously unidentified bacteriocin producers, showed biological activity. Eight propionicin-negative P127 mutants produced neither ELISA response nor biological activity. Western blot analyses of supernates detected a PLG-1 band at 9.1 kDa and two additional protein bands with apparent molecular weights of 16.2 and 27.5 kDa. PLG-1 polyclonal antibodies were used for detection of jenseniin G. PLG-1 antibodies neutralized jenseniin G activity and detected a jenseniin G-sized, 3.5 kDa peptide. Preliminary immunoprecipitation of crude preparations with PLG-1 antibodies yielded three proteins including an active 3-4 kDa band. Propionicin PLG-1 antibodies were used to screen a P. jensenii/thoenii P126 genomic expression library. Complete sequencing of a cloned insert identified by PLG-1 antibodies revealed a putative response regulator, transport protein, transmembrane protein and an open reading frame (ORF) potentially encoding jenseniin G. PCR cloning of the putative plg-1 gene yielded a 1,100 bp fragment with a 355 bp ORF encoding 118 amino acids; the deduced N-terminus was similar to the known PLG-1 N-terminus. The 118 amino acid sequence deduced from the putative plg-1 gene was larger than PLG-1 possibly due to post-translational processing. The product of the putative plg-1 gene had a calculated molecular weight of 12.8 kDa, a pI of 11.7, 14 negatively charged residues (Asp+Glu) and 24 positively charged residues (Arg+Lys). The putative plg-1 gene was expressed as an inducible fusion protein with a six-histidine residue tag. Metal affinity chromatography of the fused protein yielded a homogeneous product. The fused purified protein sequence matched the deduced putative plg-1 gene sequence. The data preliminarily suggest that both the plg-1 and jnG genes have been identified and cloned. Demonstrating that antibodies can be produced for propionicin PLG-1 and that those antibodies can be used to detect, monitor and compare activity throughout growth and purification was an important step towards monitoring PLG-1 concentrations in food systems. The unexpected but fortunate cross-reactivity of PLG-1 antibodies with jenseniin G led to selective recovery of jenseniin G by immunoprecipitation. Further refinement of this separation technique could lead to powerful affinity methods for rapid, specific separation of the two bacteriocins and thus facilitate their availability for industrial or pharmaceutical uses. Preliminary identification of genes encoding the two dairy propionibacteria bacteriocins must be confirmed; further analysis will provide means for understanding how they work, for increasing their production and for manipulating the peptides to increase their target species. Further development of these systems would contribute to basic knowledge about dairy propionibacteria and has potential for improving other industrially significant characteristics.

scholarly journals Clusters of Charged Residues at the C Terminus of MotA and N Terminus of MotB Are Important for Function of the Escherichia coli Flagellar Motor

2008 ◽  
Vol 190 (15) ◽  
pp. 5517-5521 ◽  
Author(s):  
Edan R. Hosking ◽  
Michael D. Manson
Keyword(s):  
Escherichia Coli ◽  
Flagellar Motor ◽  
Protein Levels ◽  
Partner Protein ◽  
C Terminus ◽  
N Terminus ◽  
Charged Residues ◽  
Positively Charged ◽  
Terminal Cluster

ABSTRACT MotA contains a conserved C-terminal cluster of negatively charged residues, and MotB contains a conserved N-terminal cluster of positively charged residues. Charge-altering mutations affecting these residues impair motility but do not diminish Mot protein levels. The motility defects are reversed by second-site mutations targeting the same or partner protein.

scholarly journals N Terminus of Sos1 Ras Exchange Factor: Critical Roles for the Dbl and Pleckstrin Homology Domains

1998 ◽  
Vol 18 (2) ◽  
pp. 771-778 ◽  
Author(s):  
Xiaolan Qian ◽  
William C. Vass ◽  
Alex G. Papageorge ◽  
Pieter H. Anborgh ◽  
Douglas R. Lowy
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Map Kinase ◽  
Stable Complex ◽  
Egf Receptors ◽  
Pleckstrin Homology ◽  
3T3 Cells ◽  
N Terminus ◽  
Ph Domains ◽  
Nih 3T3

ABSTRACT We have studied the functional importance of the N terminus of mouse Sos1 (mSos1), a ubiquitously expressed Ras-specific guanine nucleotide exchange factor whose C-terminal sequences bind Grb-2. Consistent with previous reports, addition of a myristoylation signal to mSos1 (MyrSos1) rendered it transforming for NIH 3T3 cells and deletion of the mSos C terminus (MyrSos1-ΔC) did not interfere with this activity. However, an N-terminally deleted myristoylated mSos1 protein (MyrSos1-ΔN) was transformation defective, although the protein was stable and localized to the membrane. Site-directed mutagenesis was used to examine the role of the Dbl and pleckstrin homology (PH) domains located in the N terminus. When mutations in the PH domain were introduced into two conserved amino acids either singly or together in MyrSos1 or MyrSos1-ΔC, the transforming activity was severely impaired. An analogous reduction in biological activity was seen when a cluster of point mutations was engineered into the Dbl domain. The mitogen-activation protein (MAP) kinase activities induced by the various Dbl and PH mutants of MyrSos1 correlated with their biological activities. When NIH 3T3 cells were transfected with a myristoylated Sos N terminus, their growth response to epidermal growth factor (EGF), platelet-derived growth factor, lysophosphatidic acid or serum was greatly impaired. The dominant inhibitory biological activity of the N terminus correlated with its ability to impair EGF-dependent activation of GTP-Ras and of MAP kinase, as well with the ability of endogenous Sos to form a stable complex with activated EGF receptors. The N terminus with mutations in the Dbl and PH domains was much less inhibitory in these biological and biochemical assays. In contrast to wild-type Sos1, nonmyristoylated versions of Sos1-ΔN and Sos1-ΔC did not form a stable complex with activated EGF receptors. We conclude that the Dbl and PH domains are critical for Sos function and that stable association of Sos with activated EGF receptors requires both the Sos N and C termini.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals Endothelial Claudin

1999 ◽  
Vol 147 (1) ◽  
pp. 185-194 ◽  
Author(s):  
Kazumasa Morita ◽  
Hiroyuki Sasaki ◽  
Mikio Furuse ◽  
Shoichiro Tsukita
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Blood Vessels ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Transmembrane Protein ◽  
Velo Cardio Facial Syndrome ◽  
The Brain ◽  
Specific Polyclonal Antibody

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6–specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell–cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face–associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell–specific component of TJ strands.

scholarly journals Epitope map of two polyclonal antibodies that recognize amyloid lesions in patients with Alzheimer's disease

1992 ◽  
Vol 282 (2) ◽  
pp. 517-522 ◽  
Author(s):  
J Ghiso ◽  
T Wisniewski ◽  
R Vidal ◽  
A Rostagno ◽  
B Frangione
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Amino Acids ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Sf9 Cells ◽  
Beta Turn ◽  
App Processing ◽  
Conformational Epitopes ◽  
N Terminus

Two synthetic peptides with sequences identical with those of fragments of the extracellular domain of the Alzheimer's-disease amyloid precursor protein (APP) were used to raise antibodies. SP28 comprises positions 597-624 of the APP695 isoform, whereas SP41 extends towards the N-terminus (amino acids 584-624) and contains the entire SP28 peptide. Using e.l.i.s.a. and inhibition experiments we identified the two beta-turn-containing segments 602-607 and 617-624 as the epitopes recognized by anti-SP41 and anti-SP28 respectively. Both antibodies immunolabelled amyloid lesions in brains from Alzheimer's-disease patients and patients with related disorders, whereas they were unreactive in control brains. However, when probed on immunoblots, anti-SP28 failed to detect full-length APP from baculovirus-infected Sf9 cells, and anti-SP41 reacted weakly compared with other anti-APP antisera. The data suggest that these antibodies are directed to conformational epitopes not existent in the native molecules but present after alternative APP processing.

scholarly journals CXCR3 Requires Tyrosine Sulfation for Ligand Binding and a Second Extracellular Loop Arginine Residue for Ligand-Induced Chemotaxis

2006 ◽  
Vol 26 (15) ◽  
pp. 5838-5849 ◽  
Author(s):  
Richard A. Colvin ◽  
Gabriele S. V. Campanella ◽  
Lindsay A. Manice ◽  
Andrew D. Luster
Keyword(s):  
Ligand Binding ◽  
Transmembrane Domain ◽  
Nk Cell ◽  
Cell Trafficking ◽  
Extracellular Loop ◽  
Ligand Interaction ◽  
Lower Affinity ◽  
Extracellular Domains ◽  
N Terminus ◽  
Charged Residues

ABSTRACT CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.

scholarly journals Design of antigen synthesis and preparation and characterization of specific and eurytopic antibodies against B-group aflatoxins

2020 ◽  
pp. 52-60
Author(s):  
Yanang Wang ◽  
Hanna Fotina
Keyword(s):  
Indirect Elisa ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Cross Reactivity ◽  
Synthesis Methods ◽  
Inhibition Concentration ◽  
Artificial Antigen ◽  
Sds Page ◽  
Sensitivity Specificity ◽  
Immune Efficacy

The aim of this study was to prepare B-group aflatoxins(BGAFs) antibody with strong specificity and good eurytopicity. According to the molecular structure and active site of aflatoxin B1 (AFB1), the BGAFs artificial antigen AFB1-BSA was prepared by 6 methods such as oxime active ester(OAE)，methylation of ammonia(MOA)，mixed anhydride(MA)，semi acetal(SA)，epoxide(EP) and enol ether derivative(EED) and identified by UV and SDS-PAGE. Polyclonal antibodies against AFB1(AFB1 pAb) were prepared by immunizing New Zealand rabbits with AFB1-BSA, and the titers of AFB1 pAb was detected by indirect ELISA, the sensitivity of AFB1 pAb was analyzed by indirect competitive ELISA(icELISA) and the specificity and eurytopicity of AFB1 pAb was analyzed by cross-reactivity(CR) test. The results showed that AFB1-BSA was synthesized successfully and the best one was OAE method among 6 synthesis methods of BGAFs artificial antigen and its conjugation ratio of AFB1 to BSA was about 8.46∶1. The immune efficacy of OAE method was the best, its AFB1 pAb had high titers of 1∶（1.28×104） by indirect ELISA, a good sensitivity with the 50% inhibition concentration(IC50) of 10.32 μg/L to AFB1 by icELISA and a high CR to AFB2 of 75.21%, AFG1 of 44.13%, AFG2 of 14.72%, AFM1 of 16.36% and AFM2 of 1.44%, respectively. In this study, AFB1 pAbs with high titer, sensitivity, specificity and eurytopicity were prepared, which laid a matter and technical foundation for the establishment of BGAFs immunoassay.

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