Influence of sodium dodecyl sulphate quality on the electrophoretic mobility of the outer membrane proteins of mucoid and non-mucoid Psedomonas aeruginosa

1983 ◽  
Vol 761 (2) ◽  
pp. 119-125 ◽  
Author(s):  
Hosmin Anwar ◽  
Peter A. Lambert ◽  
Michael R.W. Brown
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Electrophoretic Mobility ◽  
Sodium Dodecyl Sulphate ◽  
Outer Membrane Proteins ◽  
Sodium Dodecyl

Outer membrane proteins from Serratia marcescens

1993 ◽  
Vol 39 (1) ◽  
pp. 108-111 ◽  
Author(s):  
Marta Puig ◽  
Carme Fusté ◽  
Miquel Viñas
Keyword(s):  
Membrane Proteins ◽  
Serratia Marcescens ◽  
Outer Membrane ◽  
Nutrient Availability ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cultural Conditions

The outer membrane proteins (OMPs) of several strains of Serratia marcescens have been studied by sodium dodecyl sulphate – urea – polyacrylamide gel electrophoresis. Four major OMPs, named Omp1, Omp2, Omp3, and OmpA (42, 40, 39, and 37 kDa, respectively), have been visualized. The relative proportions of Omp2 and Omp3 depend on cultural conditions (temperature of incubation, osmolarity, and nutrient availability).Key words: Serratia marcescens, outer membrane proteins, porin.

scholarly journals Identification of Polymorphic Outer Membrane Proteins of Chlamydia psittaci 6BC

2001 ◽  
Vol 69 (4) ◽  
pp. 2428-2434 ◽  
Author(s):  
Regina J. Tanzer ◽  
David Longbottom ◽  
Thomas P. Hatch
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bonds ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Late Phase ◽  
Sodium Dodecyl ◽  
Chlamydia Psittaci ◽  
Developmental Cycle ◽  
Lauryl Sarcosine

ABSTRACT The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.

scholarly journals Establishing a Direct Role for the Bartonella bacilliformis Invasion-Associated Locus B (IalB) Protein in Human Erythrocyte Parasitism

2001 ◽  
Vol 69 (7) ◽  
pp. 4373-4381 ◽  
Author(s):  
Sherry A. Coleman ◽  
Michael F. Minnick
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Localization ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Amino Acid Sequence Similarity ◽  
Bartonella Bacilliformis ◽  
Sds Page

ABSTRACT The invasion-associated locus A and B genes (ialAB) ofBartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of thelacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (ρ), and cytochromeb content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.

scholarly journals Trimeric Structure of Major Outer Membrane Proteins Homologous to OmpA in Porphyromonas gingivalis

2005 ◽  
Vol 187 (3) ◽  
pp. 902-911 ◽  
Author(s):  
Keiji Nagano ◽  
Erik K. Read ◽  
Yukitaka Murakami ◽  
Takashi Masuda ◽  
Toshihide Noguchi ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Outer Membrane Proteins ◽  
Sodium Dodecyl ◽  
Membrane Integrity ◽  
Reducing Agent ◽  
Terminal Region ◽  
Mass Spectrometry Analysis ◽  
Trimeric Structure

ABSTRACT The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded β-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.

scholarly journals Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane

1982 ◽  
Vol 208 (1) ◽  
pp. 77-82 ◽  
Author(s):  
M Lindén ◽  
P Gellerfors ◽  
B D Nelson
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Charge Heterogeneity

A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.

scholarly journals Isolation and Characterization of Vibrio tubiashii Outer Membrane Proteins and Determination of a toxR Homolog

2007 ◽  
Vol 74 (3) ◽  
pp. 907-911 ◽  
Author(s):  
J. Jean-Gilles Beaubrun ◽  
M. H. Kothary ◽  
S. K. Curtis ◽  
N. C. Flores ◽  
B. E. Eribo ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Growth Conditions ◽  
Isolation And Characterization ◽  
Vibrio Tubiashii

ABSTRACT Outer membrane proteins (OMPs) expressed by Vibrio tubiashii under different environmental growth conditions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and PCR analyses. Results showed the presence of a 38- to 40-kDa OmpU-like protein and ompU gene, a maltoporin-like protein, several novel OMPs, and a regulatory toxR homolog.

scholarly journals Comparative Proteomics Study of Outer Membrane Proteins from three Pathogenic Leptospira Species used in Vaccine

2015 ◽  
Vol 5 (3) ◽  
pp. 753-760
Author(s):  
Khosrow Aghaiypour Kolyani ◽  
Rahman Shokri
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Double Diffusion ◽  
Pathogenic Leptospira ◽  
Sds Page ◽  
Antigenic Properties ◽  
Molecular Masses

 Background and Objective: Outer Membrane Proteins (OMPs) have an important role in pathogenecity and immunogenecity of Leptospira introgans. The aim of this study was chemical and immunological analysis of OMP extracted from three vaccinal strains of pathogenic Leptospira (L. canicola, L. grippotyphosa & L. sejroae hardjo) by electrophoresis and western blotting.Materials and Methods: Outer membrane enriched fractions that are insoluble in sodium N- lauryl sarcosinate isolated and studied by Sodium dodecyl sulfate – polyacrylamide gel electrophoresis. For identifying of antigenic properties of different proteins in three strains, antiserums developed in rabbit against the whole three valent vaccine and also  specific OMPs rom each of three Leptospira strains. The antiserum was used for immunological studies with immuno double diffusion and western blotting.Results: in SDS-PAGE five common protein bands with approximate molecular masses of 75,36,25,23 and 19 KDa were identified. After western blot studies identified four or five immunogenic band. In general, antiserum against L. grippotyphosa OMP has the highest ability in detecting common bands between three strains.Conclusion: Chemical and immunological comparisons between OMPs of the three strains which are being used in the commercial vaccine, provided useful documents to assess and maybe improve vaccine quality.  It indicated that OMPs are one of the major Leptospira component contributing in immunity and protectivity. Comparison between individual OMPs of each serotype revealed that L. grippotyphosa ones could contribute more in vaccine induced protectively in comparison with the other two serotypes.

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