scholarly journals Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1225
Author(s):  
Jiawen Cao ◽  
Tiantian Fan ◽  
Yanlian Li ◽  
Zhiyan Du ◽  
Lin Chen ◽  
...  
Keyword(s):  
Phage Display ◽  
Cyclic Peptide ◽  
Protein Complexes ◽  
Peptide Ligands ◽  
Phage Library ◽  
Cancer Drug ◽  
Protein Protein Interaction ◽  
Phage Display Technique ◽  
Peptide Mimic ◽  
Human Proteins

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.

A novel method of ligand peptidomics to identify peptide ligands binding to AQP2-expressing plasma membranes and intracellular vesicles of rat kidney

2008 ◽  
Vol 295 (1) ◽  
pp. F300-F309 ◽  
Author(s):  
Yu-Jung Lee ◽  
Hyo-Jung Choi ◽  
Jung-Suk Lim ◽  
Ji-Hyun Earm ◽  
Byung-Heon Lee ◽  
...  
Keyword(s):  
Phage Display ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Peptide Library ◽  
Peptide Ligands ◽  
High Affinity ◽  
Phage Display Technique ◽  
Affinity Peptide ◽  
Display Technique

Aquaporin-2 (AQP2), the vasopressin-regulated water channel in collecting duct principal cells, plays a key role in the regulation of body water balance. We aimed to isolate high-affinity peptide ligands that bind to immunoisolated AQP2-expressing plasma membrane (PM) or intracellular vesicle (ICV) preparations from rat kidney by the in vitro phage display technique. Immunoblotting revealed that AQP2 was exclusively expressed in the immunoisolated AQP2 membrane fractions (PM and ICV), compared with the nonimmunoisolated or preimmune IgG pulldown rat kidney samples. Moreover, AQP1 or H+-ATPase (B1 subunit) expression was minimal in the immunoisolated AQP2 membrane fractions, indicating the specificity of AQP2 membrane isolation. A phage peptide library based on T7 415-1b phage vector displaying CX7C was constructed. After three rounds of biopanning, seven phage clones of high frequency were selected, which showed high affinity to the AQP2-containing PM or ICV fractions compared with a nonrecombinant T7 insertless phage clone. In contrast, these phage clones showed lower affinity to H+-ATPase-containing fractions. Fluorescein-conjugated peptide labeling was associated with intracellular compartment and PM of primary cultured inner medullary collecting duct cells, relative to absent or very weak labeling with fluorescein-conjugated control peptide. Library analyses demonstrated proteins that had motifs homologous to the peptide ligands, albeit with a high probability of a random match due to short peptide sequences. In summary, we applied the in vitro phage display technique to identify high-affinity peptide ligands to AQP2-expressing membranes. Library analyses identified proteins having homologous motifs, which need to be examined for involvement in AQP2 trafficking and regulation.

Identification of a porcine TLR2-targeting peptide ligands using a cell-based phage display combined with high-throughput sequencing

2020 ◽  
Author(s):  
Su-Bin An ◽  
Seo-Ho Oh ◽  
Jun-Yeong Lee ◽  
Kwang-Hwan Choi ◽  
Chang-Kyu Lee ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Oral Route ◽  
Peptide Ligands ◽  
Mucosal Vaccine ◽  
Cell Targeting ◽  
M Cell ◽  
Phage Display Technique

Abstract M cell targeting is one of the critical issues to develop efficient mucosal vaccine design. In this study, peptide ligands with high affinity to porcine TLR2, which is highly expressed in M cells and play an important role in mucosal immune responses in pigs, were identified through the cell-based phage display technique combined with high-throughput sequencing. A random phage-peptide library was applied to the porcine TLR2 overexpressing cell line and total 85, 557 unique peptide sequences were identified from approximately 9.0 × 107 reads after three rounds of both subtractive and non-subtractive biopanning via high-throughput sequencing. Among the unique sequences, three candidate peptide sequences, NAGHLSQ, VPSKPGL, and RANLDGQ, were selected based on their abundance in the third round of biopanning. Consequently, NAGHLSQ showed the highest affinity exclusively to porcine TLR2 compared with other candidates and its binding mechanism was inferred to be directly associated with ligand binding site of the TLR2 through the in vitro competitive analysis. The peptide identified in this research could be used in development of effective porcine mucosal vaccine as an M cell targeting moiety to enhance the transport of antigens into the Peyer's patch via oral route.

Development and Application of Computational Methods in Phage Display Technology

2020 ◽  
Vol 26 (42) ◽  
pp. 7672-7693 ◽  
Author(s):  
Bifang He ◽  
Anthony Mackitz Dzisoo ◽  
Ratmir Derda ◽  
Jian Huang
Keyword(s):  
Phage Display ◽  
Computational Methods ◽  
Peptide Ligands ◽  
Next Generation ◽  
Phage Display Technology ◽  
Next Generation Sequencing Technology ◽  
Screening Experiments ◽  
Display Technology ◽  
Comprehensive Search ◽  
Generation Sequencing

Background: Phage display is a powerful and versatile technology for the identification of peptide ligands binding to multiple targets, which has been successfully employed in various fields, such as diagnostics and therapeutics, drug-delivery and material science. The integration of next generation sequencing technology with phage display makes this methodology more productive. With the widespread use of this technique and the fast accumulation of phage display data, databases for these data and computational methods have become an indispensable part in this community. This review aims to summarize and discuss recent progress in the development and application of computational methods in the field of phage display. Methods: We undertook a comprehensive search of bioinformatics resources and computational methods for phage display data via Google Scholar and PubMed. The methods and tools were further divided into different categories according to their uses. Results: We described seven special or relevant databases for phage display data, which provided an evidence-based source for phage display researchers to clean their biopanning results. These databases can identify and report possible target-unrelated peptides (TUPs), thereby excluding false-positive data from peptides obtained from phage display screening experiments. More than 20 computational methods for analyzing biopanning data were also reviewed. These methods were classified into computational methods for reporting TUPs, for predicting epitopes and for analyzing next generation phage display data. Conclusion: The current bioinformatics archives, methods and tools reviewed here have benefitted the biopanning community. To develop better or new computational tools, some promising directions are also discussed.

scholarly journals Phage display demonstrates durable differences in serological profile by route of inoculation in primary infections of non-human primates with Dengue Virus 1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jayant V. Rajan ◽  
Michael McCracken ◽  
Caleigh Mandel-Brehm ◽  
Greg Gromowski ◽  
Simon Pollett ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Phage Display ◽  
Denv Infection ◽  
Phage Library ◽  
Humoral Immune ◽  
Linear Peptide ◽  
Humoral Immune Responses ◽  
High Resolution Map ◽  
Domain Iii ◽  
Inoculation Route

AbstractNatural dengue virus (DENV) infections occur by mosquito bite but how the inoculation route affects the humoral immune response is unknown. We serologically profiled 20 non-human primates (NHP) from a prior study of DENV1 infection where animals were inoculated by mosquito (N = 10) or subcutaneous injection (N = 10). Using a comprehensive, densely tiled and highly redundant pan-flavivirus programmable phage library containing 91,562 overlapping 62 amino acid peptides, we produced a high-resolution map of linear peptide sequences enriched during DENV seroconversion. Profiles in mosquito-inoculated and subcutaneously-inoculated animals were similar up to 90 days after primary infection, but diverged at 1 year with differences in sero-reactivity in the Envelope (E; residues 215–406; p < 0.08), and Nonstructural-3 (NS3; residues 549–615; p < 0.05) proteins in mosquito-inoculated versus subcutaneously-inoculated animals. Within the E protein, residues 339–384 in domain III accounted for > 99% of the observed sero-reactivity difference. Antibody breadth did not vary by mode of inoculation. The differential reactivity to E domain III seen by phage display validated orthogonally by ELISA, but did not correlate with late neutralization titers. Serological profiling of humoral immune responses to DENV infection in NHP by programmable phage display demonstrated durable differences in sero-reactivity by route of inoculation.

scholarly journals Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

1994 ◽  
Vol 124 (3) ◽  
pp. 373-380 ◽  
Author(s):  
E Koivunen ◽  
B Wang ◽  
E Ruoslahti
Keyword(s):  
Phage Display ◽  
Cyclic Peptide ◽  
Cell Attachment ◽  
Phage Display Library ◽  
Cell Binding ◽  
Rgd Peptides ◽  
Beta 1 Integrin ◽  
Alpha V Beta 3 ◽  
Integrin Ligand ◽  
Specific Ligand

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.

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