Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori

2015 ◽  
Vol 21 (9) ◽  
pp. 710-716 ◽  
Author(s):  
Yumiko Hayakawa ◽  
Mitsuhiro Matsuno ◽  
Makoto Tanaka ◽  
Akihiro Wada ◽  
Koichiro Kitamura ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Complementary Dna ◽  
High Affinity ◽  
Vacuolating Toxin ◽  
Selection Of

In vitro selection of high affinity HspR-binding sites within the genome of Helicobacter pylori

Gene ◽  
2002 ◽  
Vol 283 (1-2) ◽  
pp. 63-69 ◽  
Author(s):  
Isabel Delany ◽  
Gunther Spohn ◽  
Rino Rappuoli ◽  
Vincenzo Scarlato
Keyword(s):  
Helicobacter Pylori ◽  
Binding Sites ◽  
In Vitro Selection ◽  
High Affinity ◽  
Selection Of

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Precise selection of aptamers targeting PD-L1 positive small extracellular vesicles on magnetic chips

2021 ◽  
Vol 57 (29) ◽  
pp. 3555-3558
Author(s):  
Hao-Yu Dong ◽  
Qi-Hui Xie ◽  
Dai-Wen Pang ◽  
Gang Chen ◽  
Zhi-Ling Zhang
Keyword(s):  
Extracellular Vesicles ◽  
High Affinity ◽  
Selection Of

High affinity aptamers that target small extracellular vesicles displaying PD-L1 in its natural conformation were successfully selected.

In Vitro Selection of Oligonucleotide Acid Aptamers against Pathogenic Vibrio Alginolyticus by SELEX

2010 ◽  
Vol 439-440 ◽  
pp. 1456-1462 ◽  
Author(s):  
Jiang Zheng ◽  
Yu Bao Li ◽  
Jia Xiang Li ◽  
Jun Wang ◽  
Yong Quan Su
Keyword(s):  
Secondary Structures ◽  
Magnetic Bead ◽  
Vibrio Alginolyticus ◽  
Pathogenic Microorganism ◽  
Asymmetric Pcr ◽  
High Affinity ◽  
Amplification Method ◽  
Pathogenic Vibrio ◽  
Selection Of

Detection of pathogenic microorganism is very necessary in the control of infectious disease prevailing in aquiculture animals. However, most of the present techniques can not meet the need of the quick field inspection. Systematic evolution of ligands by exponential enrichment (SELEX) is a new molecular recognition way for generating high affinity oligonucleotide acid aptamers, a new nucleotide acid material, which have been widely used in the detections of proteins, cells and so on. In the present paper, the technology was applied to select the high affinity aptamers against pathogenic microorganism Vibrio alginolyticus, which could be used for the rapid field detection of the microorganism. Based on the designment of the ssDNA library of 76 nucleotide acids with 35-base random region, the SELEX system for the selection of the high affinity aptamers against Vibrio alginolyticus was established. In the SELEX system, asymmetric PCR was proved to be a better amplification method for the ssDNA library than the reported affinity magnetic bead method, and the corresponding parameters of the asymmetric PCR were also studied and optimized. The affinity of the final ssDNA library increased by nearly 200% compared with the original library. Cloning and sequencing of the final ssDNA library showed that there were at least two kinds of ssDNAs with different length in the affinity ssDNA library: one was 76 bases, another was 149 bases. Simulation of the secondary structures showed that the secondary structures of the two fragments were different greatly, suggesting that the two fragments could bind to different sites of V. alginolyticus surface.

scholarly journals Helicobacter pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity

2015 ◽  
Vol 197 (11) ◽  
pp. 1886-1892 ◽  
Author(s):  
Jennifer Tsang ◽  
Takanori Hirano ◽  
Timothy R. Hoover ◽  
Jonathan L. McMurry
Keyword(s):  
Helicobacter Pylori ◽  
Response Regulator ◽  
Regulatory Protein ◽  
Kinase Domain ◽  
Optical Biosensing ◽  
Content Type ◽  
High Affinity ◽  
Flagellar Assembly ◽  
Flagellar Biogenesis

ABSTRACTFlagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. InHelicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoN-dependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Deletion of its N-terminal cytoplasmic sequence dramatically decreased expression of two RpoN-dependent genes,flaBandflgE. Optical biosensing demonstrated a high-affinity interaction between FlgS and a peptide consisting of residues 1 to 25 of FlhA (FlhANT). TheKD(equilibrium dissociation constant) was 21 nM and was characterized by fast-on (kon= 2.9 × 104M−1s−1) and slow-off (koff= 6.2 × 10−4s−1) kinetics. FlgS did not bind peptides consisting of smaller fragments of the FlhANTsequence. Analysis of binding to purified fragments of FlgS demonstrated that the C-terminal portion of the protein containing the kinase domain binds FlhANT. FlhANTbinding did not stimulate FlgS autophosphorylationin vitro, suggesting that FlhA facilitates interactions between FlgS and other structures required to stimulate autophosphorylation.IMPORTANCEThe high-affinity binding of FlgS to FlhA characterized in this study points to an additional role for FlhA in flagellar assembly. Beyond its necessity for type III secretion, the N-terminal cytoplasmic sequence of FlhA is required for RpoN-dependent gene expression via interaction with the C-terminal kinase domain of FlgS.

scholarly journals Evolved polymerases facilitate selection of fully 2′-OMe-modified aptamers

2017 ◽  
Vol 8 (12) ◽  
pp. 8179-8182 ◽  
Author(s):  
Zhixia Liu ◽  
Tingjian Chen ◽  
Floyd E. Romesberg
Keyword(s):  
Dna Polymerases ◽  
High Affinity ◽  
Selection Of

Evolved DNA polymerases are used in selections with fully 2′-OMe modified libraries to identify aptamers with high affinity for HNE.

Helicobacter pylori vacuolating toxin VacA

2003 ◽  
pp. 60-75
Author(s):  
Cesare Montecucco ◽  
Francesco Tombola ◽  
Emanuele Papini ◽  
Lucantonio Debellis ◽  
Mario Zoratti
Keyword(s):  
Helicobacter Pylori ◽  
Vacuolating Toxin
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