scholarly journals Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria

2020 ◽  
Vol 8 (5) ◽  
pp. 780
Author(s):  
Armando Navarro ◽  
Delia Licona-Moreno ◽  
Alejandro Monsalvo-Reyes ◽  
Ulises Hernández-Chiñas ◽  
Carlos A. Eslava-Campos
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Amino Acid Sequences ◽  
Serum Samples ◽  
E Coli ◽  
Peptide Mimotopes ◽  
Human Sera ◽  
Shared Epitopes

Background: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. Aim: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. Methods: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. Results: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. Conclusion: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

Immunogenic peptide mimotopes from an epitope of Escherichia coli O157 LPS

2016 ◽  
Vol 473 (21) ◽  
pp. 3791-3804 ◽  
Author(s):  
Armando Navarro ◽  
Ulises Hernández-Chiñas ◽  
Delia Licona-Moreno ◽  
Edgar Zenteno ◽  
Alejandro Cravioto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Escherichia Coli O157 ◽  
Serum Samples ◽  
Haemolytic Uremic Syndrome ◽  
E Coli ◽  
Positive Reactivity ◽  
Peptide Mimotopes ◽  
Uremic Syndrome

Escherichia coli O157:H7 is a subtype of Shiga toxin-producing E. coli that is associated with haemorrhagic colitis and haemolytic uremic syndrome (HUS). Studies of populations in endemic areas have reported that the presence of specific antibodies against the O157 lipopolysaccharide (LPS) is associated with a lower incidence of diarrhoea and HUS. Phage display and IgG anti-O157 LPS antibodies were used in the present study to select peptide mimotopes of O157 LPS expressed in protein III of the M13 phage. Synthetic peptides (SP) were designed using the derived amino acid sequences obtained from DNA nucleotides of 63 selected phagotopes. The LxP/YP/SxL motif was identified in five of the phagotope amino acid sequences. Antibody responses against the phagotopes and their corresponding SPs were evaluated. SP12, one of the designed SP, induced the production of antibodies against the homologous peptide (1:800) and O157 LPS (1:200). The specificity of anti-SP12 antiserum was confirmed by analyzing its response to SP3, an SP with a different amino acid sequence than that of SP12, as well as against an E. coli LPS different from O157. Competitive studies with SP12 and O157 LPS showed a significant decrease in anti-SP12 and anti-LPS O157 antiserum responses against SP12 and O157 LPS, respectively. Eighteen (82%) of the 22 human serum samples with positive reactivity against E. coli O157 LPS reacted with SP12 SP (cut-off >0.4). These results support the idea that SP12 is an immunogenic mimotope of O157 LPS.

BCRs of Mutated and Unmutated CLL Bind Peptides with Distinct Sequence Features: Evidence from Phage Display Libraries.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1118-1118
Author(s):  
Manuela Woelfle ◽  
Till Seiler ◽  
Rosa Catera ◽  
Hartmut Dohner ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Specific Binding ◽  
Phage Display Library ◽  
Amino Acid Sequences ◽  
Phage Display Technology ◽  
Ligand Interactions ◽  
Distinct Sequence ◽  
Peptide Phage Display

Abstract In CLL, the use of specific IgV genes to code the clone’s BCR is non-random and there is an apparent selection for particular genetic and amino acid structures that can be shared by different patients, supporting the hypothesis that antigenic stimulation influences the development and course of CLL. As the binding specificities of the BCR are largely unknown, a vast variety of antigens may affect the BCRs and defined antigens have yet to be identified. Therefore, we used peptide phage display technology to identify ligands for CLL BCRs. BCRs from 2 IgVH unmutated (U-CLL) and 3 mutated (M-CLL) patients were expressed as IgG1 mAbs and used to probe a 12-mer peptide phage display library. In each case, after 3 rounds of selection, we isolated ligands reactive with the CLL mAbs. For the 3 M-CLL mAbs, phage clones carrying peptide inserts with conserved consensus motifs were found. Specificity of the BCR-ligand interactions was demonstrated in direct and indirect ELISA, since selected phage clones and synthetic peptides bound to their respective M-CLL mAb but not to other M-CLL mAbs. Variation of the amino acid sequence of the synthetic peptides significantly altered their reactivity with the corresponding M-CLL mAb. Furthermore, synthetic peptides were bound only by the proper mAb/BCR, but not by mAbs of other M-CLL or U-CLL patients with BCRs comprised of different IgVH genes, supporting the hypothesis that BCRs of M-CLL recognize a defined epitope. In contrast, the mAbs from 2 U-CLL cases did not select phages bearing a consensus motif. Rather these U-CLL mAbs bound multiple phages expressing the same 12-mer peptides, although these differed in sequence between the two U-CLL cases tested. Furthermore, 2 separate selection procedures using 1 U-CLL mAb isolated multiple phage bearing the same 12-mer sequence on each occasion as well as another set of phages with a completely distinct sequence in 1 of 2 selections. ELISAs demonstrated specific binding of all phage clones and of the synthetic peptides by the U-CLL mAbs. Despite this level of specificity, the 2 U-CLL mAbs also reacted with peptides isolated from panning with other CLL mAbs, thereby displaying considerable polyreactivity. Rather than binding only one distinct epitope, mAbs from U-CLL appear capable of interacting with multiple, unrelated structures. Finally, one of the peptides isolated with an U-CLL mAb was bound by all of the CLL mAbs tested, including those from M-CLL cases; therefore this target is antigenically “polyreactive”. Thus, phage display is a feasible approach to identify specific ligands for CLL BCRs. The two classes of BCRs in M-CLL and U-CLL show substantially different binding properties - the former binding shared amino acid motifs and the latter binding multiple ligands of distinct and identical 12-mer amino acid sequences. These peptides can be used to analyze more precisely the binding sites of CLL BCRs as well as the consequences that ensue after BCR crosslinking, and they might help develop BCR-specific therapeutic agents.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

scholarly journals Induction of neonatal lupus in pups of mice immunized with synthetic peptides derived from amino acid sequences of the serotoninergic 5-HT4 receptor

2001 ◽  
Vol 31 (2) ◽  
pp. 573-579 ◽  
Author(s):  
Pierre Eftekhari ◽  
Jean-Christophe Roegel ◽  
Frank Lezoualc'h ◽  
Rodolphe Fischmeister ◽  
Jean-Louis Imbs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
Amino Acid Sequences ◽  
Neonatal Lupus

scholarly journals Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Xiao Teng Ching ◽  
Yee Ling Lau ◽  
Mun Yik Fong ◽  
Veeranoot Nissapatorn ◽  
Hemah Andiappan
Keyword(s):  
Western Blot ◽  
Affinity Purification ◽  
Expression System ◽  
Economic Loss ◽  
Serum Samples ◽  
Serological Tests ◽  
Human Patient ◽  
Prokaryotic Expression System ◽  
Human Sera ◽  
Lysate Antigens

Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).

Cefotaxime-Resistant EnterobacteriaceaeIsolates from a Hospital in Warsaw, Poland: Identification of a New CTX-M-3 Cefotaxime-Hydrolyzing β-Lactamase That Is Closely Related to the CTX-M-1/MEN-1 Enzyme

1998 ◽  
Vol 42 (4) ◽  
pp. 827-832 ◽  
Author(s):  
Marek Gniadkowski ◽  
Ines Schneider ◽  
Andrzej Pal/ucha ◽  
Renate Jungwirth ◽  
Barbara Mikiewicz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Clinical Laboratory ◽  
Amino Acid Sequences ◽  
Hospital Environment ◽  
M Gene ◽  
E Coli ◽  
Men 1 ◽  
Long Time ◽  
Dna Restriction ◽  
Tract Infections

ABSTRACT A group of cefotaxime-resistant Citrobacter freundiiand Escherichia coli isolates were collected by a clinical laboratory in a hospital in Warsaw, Poland, in July 1996. Detailed analysis has shown that all of these produced a β-lactamase (pI, 8.4) belonging to the CTX-M family, one of the minor extended-spectrum β-lactamase families with a strong cefotaxime-hydrolyzing activity. Sequencing has revealed that C. freundii isolates produced a new CTX-M-3 enzyme which is very closely related to the CTX-M-1/MEN-1 β-lactamase, sporadically identified in Europe over a period of 6 years. Amino acid sequences of these two β-lactamases differ at four positions: Val77Ala, Asp114Asn, Ser140Ala, and Asn288Asp (the first amino acid of each pair refers to CTX-M-1/MEN-1 and second refers to CTX-M-3). The partial sequence of the E. coli CTX-M gene was identical to the corresponding region ofbla CTX-M-3, but a transconjugant of theE. coli isolate expressed higher levels of resistance to β-lactams than did C. freundii transconjugants. These resistance differences correlated with differences in plasmid DNA restriction patterns. Our results suggest that CTX-M genes have been spread among different species of the familyEnterobacteriaceae in the hospital and that the CTX-M-3-expressing C. freundii strain causing routine urinary tract infections has been maintained for a relatively long time in the hospital environment.

Subunit II (b') and Not Subunit I (b) of Photosynthetic ATP Synthases is Equivalent to Subunit b of the ATP Synthases from Nonphotosynthetic Eubacteria. Evidence for a New Assignment of b-Type F0 Subunits

1997 ◽  
Vol 52 (11-12) ◽  
pp. 789-798 ◽  
Author(s):  
Hans-Jürgen Tiburzy ◽  
Richard J. Berzborn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Structural Feature ◽  
Atp Synthase ◽  
Primary Structure ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
E Coli ◽  
Subunit B ◽  
Atp Synthases

Abstract Subunit I of chloroplast ATP synthase is reviewed until now to be equivalent to subunit b of Escherichia coli ATP synthase, whereas subunit II is suggested to be an additional subunit in photosynthetic ATP synthases lacking a counterpart in E. coli. After publication of some sequences of subunits II a revision of this assignment is necessary. Based on the analysis of 51 amino acid sequences of b-type subunits concerning similarities in primary structure, iso­electric point and a discovered discontinuous structural feature, our data provide evidence that chloroplast subunit II (subunit b' of photosynthetic eubacteria) and not chloroplast subunit I (subunit b of photosynthetic eubacteria) is the equivalent of subunit b of nonphoto­ synthetic eubacteria, and therefore does have a counterpart in e.g. E. coli. In consequence, structural features essential for function should be looked for on subunit II (b').

Sign in / Sign up

Export Citation Format

Share Document