scholarly journals Isolation of a Treponema pallidum gene encoding immunodominant outer envelope protein P6, which reacts with sera from patients at different stages of syphilis.

1986 ◽  
Vol 164 (4) ◽  
pp. 1160-1170 ◽  
Author(s):  
K M Peterson ◽  
J B Baseman ◽  
J F Alderete
Keyword(s):  
Envelope Protein ◽  
Treponema Pallidum ◽  
Surface Protein ◽  
Plasmid Vector ◽  
Transformed Cells ◽  
Outer Envelope ◽  
Gene Encoding ◽  
Recombinant Phage ◽  
E Coli ◽  
Outer Membranes

A phage directing the synthesis of an abundant 45-kD Treponema pallidum surface protein was isolated from an EMBL-4 bacteriophage lambda library of T. pallidum DNA. The recombinant phage was identified using an mAb that was directed toward an immunodominant, outer envelope T. pallidum protein designated P6. The recombinant P6 protein possessed the same mol mass as the native treponemal antigen detected from total T. pallidum protein preparations, confirming the cloning of the structural gene for this molecule. Furthermore, E. coli was transformed by a 4.5-kb Eco RI lambda insert fragment subcloned into the plasmid vector pUC19. These transformed cells expressed and translocated the 45-kD protein to their outer membranes. Finally, all sera from patients with different stages of syphilis (primary, secondary, and latent) contained antibody reactive to this protein.

Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea

2000 ◽  
Vol 28 (6) ◽  
pp. 967-969 ◽  
Author(s):  
M. N. A. Mandal ◽  
I. M. Santha ◽  
M. L. Lodha ◽  
S. L. Mehta
Keyword(s):  
Brassica Juncea ◽  
Southern Hybridization ◽  
Genomic Library ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Plasmid Vector ◽  
Phage Clone ◽  
Carrier Protein ◽  
Gene Encoding ◽  
Recombinant Phage

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in λEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone λ5.12 after a series of screening, sub-cloning and Southern hybridization.

scholarly journals Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

2001 ◽  
Vol 183 (16) ◽  
pp. 4702-4708 ◽  
Author(s):  
Stéphane Benoit ◽  
James E. Posey ◽  
Matthew R. Chenoweth ◽  
Frank C. Gherardini
Keyword(s):  
Treponema Pallidum ◽  
Maximum Growth ◽  
Biochemical Properties ◽  
Maximum Activity ◽  
Phosphoglycerate Mutase ◽  
Heat Sensitivity ◽  
Dependent Manner ◽  
Gene Encoding ◽  
E Coli ◽  
Key Enzyme

ABSTRACT In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP inT. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn2+ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.

scholarly journals Multiple Domains of the Jaagsiekte Sheep Retrovirus Envelope Protein Are Required for Transformation of Rodent Fibroblasts

2004 ◽  
Vol 78 (19) ◽  
pp. 10479-10489 ◽  
Author(s):  
Andrew Hofacre ◽  
Hung Fan
Keyword(s):  
Envelope Protein ◽  
Transmembrane Protein ◽  
Surface Protein ◽  
Cytoplasmic Tail ◽  
Deletion Mutants ◽  
Gene Encoding ◽  
Jaagsiekte Sheep Retrovirus ◽  
Confocal Immunofluorescence Microscopy ◽  
Predicted Signal Peptide ◽  
Nih 3T3

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma. We previously showed that the gene encoding JSRV envelope protein (Env) appears to function as an oncogene, since it can transform mouse NIH 3T3 cells. The cytoplasmic tail of the Env transmembrane protein (TM) is necessary for the transformation. However, previous experiments did not exclude the involvement of the Env surface protein (SU) in transformation. In this study, we created a series of nested deletion mutants through the SU domain and assessed their ability to transform rodent fibroblasts. All SU deletion mutants downstream of the predicted signal peptide were unable to transform murine NIH 3T3 or rat 208F cells. Transport to the plasma membrane of selected deleted Env proteins was confirmed by confocal immunofluorescence microscopy of hemagglutinin-tagged versions. Additional sequential SU deletion mutants lacking 50-amino-acid (aa) blocks throughout SU also were unable to transform. Furthermore, minimal insertion mutants of two amino acids (Leu/Gln) at various positions in SU also abolished transformation. These data indicate that domains in SU facilitate efficient JSRV transformation. This could reflect a necessity of SU for appropriate configuration of the Env protein or independent activation by SU of a signaling pathway necessary for transformation. Complementation between SU and TM mutants for transformation supported the latter hypothesis. Cotransfection with ΔGP Y590F (mutant in the TM cytoplasmic tail) with ΔGP SUΔ103-352 (lacking most of SU) resulted in efficient transformation. The resulting transformants showed evidence for the presence and expression of both mutant plasmids.

scholarly journals Use of a Monoclonal Antibody against anEscherichia coli O26 Surface Protein for Detection of Enteropathogenic and Enterohemorrhagic Strains

1999 ◽  
Vol 6 (4) ◽  
pp. 610-614 ◽  
Author(s):  
Paul Kerr ◽  
Hywel Ball ◽  
Bernard China ◽  
Jacques Mainil ◽  
David Finlay ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Outer Membrane Proteins ◽  
Surface Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enteric Disease ◽  
Gene Encoding ◽  
E Coli ◽  
Diagnostic Potential ◽  
Elisa Format ◽  
Enteric Infections

ABSTRACT A monoclonal antibody (MAb) was obtained from a mouse immunized with solubilized outer membrane proteins extracted from a bovine enterohemorrhagic strain of Escherichia coli (EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26 eae- and VT-negative strain, or O157 eae- and VT-positive strains. The MAb was used in a sandwich enzyme-linked immunosorbent assay (ELISA) format to screen strains from animal and human sources, and all reactive strains were characterized for the presence of eaeand the gene encoding VT factors by PCR. The antigen was detected in a group of strains containing a high proportion of O26, the majority of which were eae positive with or without VT; these were isolated mostly from animal enteritis cases but included a small number of human enteric isolates. Nonreactors includedeae-positive (with or without VT) O157 strains and one O26 strain. In a survey of mixed cultures from both animal and human enteric disease, ELISA-positive reactions were obtained from 7.1 to 11.2% of samples from bovine, porcine, ovine, and human sources. The two human O8 and ten animal O26 ELISA-reactive pure strains obtained from these samples contained six eae- and/or VT-positive strains; the other six strains lost their ELISA positivity following storage at −70°C, after which none were found to contain eithereae or VT factors. The association of the antigen detected by the MAb with significant enteropathogenic E. coli and EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.

Regulation of the synthesis of surface protein in the cell cycle of e. coli b/r

Cell ◽  
1979 ◽  
Vol 18 (2) ◽  
pp. 287-296 ◽  
Author(s):  
A. Boyd ◽  
I.B. Holland
Keyword(s):  
Cell Cycle ◽  
Surface Protein ◽  
E Coli

scholarly journals Cloning of Salmonella enterica Serovar Enteritidis Fimbrial Protein SefA as a Surface Protein in Escherichia coli Confers the Ability To Attach to Eukaryotic Cell Lines

2009 ◽  
Vol 75 (20) ◽  
pp. 6622-6625 ◽  
Author(s):  
Douglas L. Rank ◽  
Mahdi A. Saeed ◽  
Peter M. Muriana
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Expression Vector ◽  
Surface Protein ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Western Blot Assay ◽  
E Coli ◽  
Fimbrial Protein

ABSTRACT The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

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