scholarly journals A Phage-Compatible Strategy to Access Macrocyclic Peptides Featuring Asymmetric Molecular Scaffolds as Cyclization Units

2021 ◽  
Author(s):  
Titia Rixt Oppewal ◽  
Johan Hekelaar ◽  
Clemens Mayer
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Cysteine Residue ◽  
Molecular Scaffolds ◽  
Noncanonical Amino Acids ◽  
Disulfide Formation ◽  
Macrocyclic Peptides ◽  
Selection Of

The cyclization of peptides appended onto proteins or whole bacteriophages is typically achieved via disulfide formation, the use of symmetric crosslinkers or the incorporation of noncanonical amino acids. Unfortunately, neither of these strategies is amenable toward generating libraries for the selection of macrocyclic peptides (MPs) akin to those found in nature, which often feature asymmetric molecular scaffolds as cyclization units that improve binding to their targets. To meet this challenge, we present an efficient two-step strategy to access MPs via the programmed modification of a unique cysteine residue and an N-terminal amine. We demonstrate that this approach yields MPs featuring asymmetric cyclization units from both synthetic peptides and when linear precursors are appended onto a phage-coat protein. Given that the employed conditions are compatible with phage display protocols, our work paves the way for the selection of natural-product-like MPs from randomized peptide sequences by phage display.

scholarly journals A Phage-Compatible Strategy to Access Macrocyclic Peptides Featuring Asymmetric Molecular Scaffolds as Cyclization Units

2021 ◽  
Author(s):  
Titia Rixt Oppewal ◽  
Johan Hekelaar ◽  
Clemens Mayer
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Cysteine Residue ◽  
Molecular Scaffolds ◽  
Noncanonical Amino Acids ◽  
Disulfide Formation ◽  
Macrocyclic Peptides ◽  
Selection Of

The cyclization of peptides appended onto proteins or whole bacteriophages is typically achieved via disulfide formation, the use of symmetric crosslinkers or the incorporation of noncanonical amino acids. Unfortunately, neither of these strategies is amenable toward generating libraries for the selection of macrocyclic peptides (MPs) akin to those found in nature, which often feature asymmetric molecular scaffolds as cyclization units that improve binding to their targets. To meet this challenge, we present an efficient two-step strategy to access MPs via the programmed modification of a unique cysteine residue and an N-terminal amine. We demonstrate that this approach yields MPs featuring asymmetric cyclization units from both synthetic peptides and when linear precursors are appended onto a phage-coat protein. Given that the employed conditions are compatible with phage display protocols, our work paves the way for the selection of natural-product-like MPs from randomized peptide sequences by phage display.

A novel phage display vector for selection of target-specific peptides

2020 ◽  
Vol 33 ◽  
Author(s):  
Alex Chang ◽  
Joey P Ting ◽  
Alfonso Espada ◽  
Howard Broughton ◽  
Manuel Molina-Martin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Phage Display ◽  
Copy Number ◽  
Wild Type ◽  
Phage Display Technology ◽  
Successful Isolation ◽  
Display Technology ◽  
Therapeutic Peptides ◽  
Selection Of

Abstract Intrinsic low display level of polypeptides on phage is a fundamental and limiting hurdle in successful isolation of target-specific binders by phage display technology. To circumvent this challenge, we optimized the copy number of peptides displayed on the phage surface using type 33 phage vector. We randomized the first 67 amino acids of the wild type PIII to identify mutants that would result in its reduced expression. Consequently, the display level was improved by 30-fold due to higher incorporation of the synthetic PIII–peptide fusion protein on the phage surface. Utilization of this novel phage vector should provide a solid basis for the discovery of therapeutic peptides.

Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC

2019 ◽  
Vol 8 (1) ◽  
pp. 24-31
Author(s):  
Chol-Jin Kim ◽  
Sunll Choe ◽  
Kum-Chol Ri ◽  
Chol-Ho Kim ◽  
Hyon-Gwang Li ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Phage Display ◽  
Affinity Purification ◽  
Phage Library ◽  
Amino Acid Residues ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Selection Of

Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.

scholarly journals Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Johannes H. Urban ◽  
Markus A. Moosmeier ◽  
Tobias Aumüller ◽  
Marcus Thein ◽  
Tjibbe Bosma ◽  
...  
Keyword(s):  
Coat Protein ◽  
Phage Display ◽  
Carboxy Terminus ◽  
Minor Coat Protein ◽  
Selection Of

scholarly journals Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases

2021 ◽  
Author(s):  
Jonathan Tremblay ◽  
Marie-Claire Goulet ◽  
Juan Vorster ◽  
Charles Goulet ◽  
Dominique Michaud
Keyword(s):  
Amino Acids ◽  
Phage Display ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Structural Elements ◽  
New Approach ◽  
Digestive Proteases ◽  
Highly Active ◽  
Plant Cystatin ◽  
Selection Of

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approaches typically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as donors of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model recipient inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

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.

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

2019 ◽  
Vol 19 (22) ◽  
pp. 1952-1961 ◽  
Author(s):  
J.C. Sobrinho ◽  
A.F. Francisco ◽  
R. Simões-Silva ◽  
A.M. Kayano ◽  
J.J. Alfonso Ruiz Diaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Synthetic Peptides ◽  
Cell Damage ◽  
Neutralization Assay ◽  
Phospholipase Activity ◽  
Phospholipases A2 ◽  
Catalytically Active ◽  
Bothrops Atrox

Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

Sign in / Sign up

Export Citation Format

Share Document