Phage Display: A Powerful Technology for the Generation of High-Specificity Affinity Reagents from Alternative Immune Sources

AbstractIntroductionThe generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.MethodsUsing a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.Results10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.ConclusionsOur data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach should be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

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A phage display-based strategy for the de novo creation of disulfide-constrained and isomer-free bicyclic peptide affinity reagents

Chemical Communications ◽

10.1039/c7cc09142g ◽

2018 ◽

Vol 54 (32) ◽

pp. 4029-4032 ◽

Cited By ~ 8

Author(s):

Mirao Zha ◽

Ping Lin ◽

Hongwei Yao ◽

Yibing Zhao ◽

Chuanliu Wu

Keyword(s):

Phage Display ◽

Genetic Code ◽

De Novo ◽

Screening Strategy ◽

Peptide Ligands ◽

Disulfide Bridges ◽

Chemical Modifications ◽

Affinity Reagents ◽

Peptide Affinity

We report a phage-screening strategy for the development of bicyclic peptide ligands constrained with two sterically different and isomerically forbidden noncanonical disulfide bridges without elaborate chemical modifications and recourses to genetic code reprogramming.

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High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation

The FASEB Journal ◽

10.1096/fasebj.2018.32.1_supplement.544.16 ◽

2018 ◽

Vol 32 (S1) ◽

Author(s):

Loretta Yang ◽

Shengcheng Wu ◽

J. Christopher Cooper ◽

Mallory K. Paul ◽

Amanda L. Cummings ◽

...

Keyword(s):

High Specificity ◽

Affinity Reagents

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DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing

Scientific Reports ◽

10.1038/s41598-020-77221-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Claudia Kolm ◽

Isabella Cervenka ◽

Ulrich J. Aschl ◽

Niklas Baumann ◽

Stefan Jakwerth ◽

...

Keyword(s):

State Of The Art ◽

Selection Process ◽

High Specificity ◽

Cost Effective ◽

Cell Labelling ◽

Dna Aptamers ◽

Bacterial Cells ◽

Combined Approach ◽

Affinity Reagents ◽

Assessment Techniques

AbstractDNA aptamers generated by cell-SELEX against bacterial cells have gained increased interest as novel and cost-effective affinity reagents for cell labelling, imaging and biosensing. Here we describe the selection and identification of DNA aptamers for bacterial cells using a combined approach based on cell-SELEX, state-of-the-art applications of quantitative real-time PCR (qPCR), next-generation sequencing (NGS) and bioinformatic data analysis. This approach is demonstrated on Enterococcus faecalis (E. faecalis), which served as target in eleven rounds of cell-SELEX with multiple subtractive counter-selections against non-target species. During the selection, we applied qPCR-based analyses to evaluate the ssDNA pool size and remelting curve analysis of qPCR amplicons to monitor changes in pool diversity and sequence enrichment. Based on NGS-derived data, we identified 16 aptamer candidates. Among these, aptamer EF508 exhibited high binding affinity to E. faecalis cells (KD-value: 37 nM) and successfully discriminated E. faecalis from 20 different Enterococcus and non-Enterococcus spp. Our results demonstrate that this combined approach enabled the rapid and efficient identification of an aptamer with both high affinity and high specificity. Furthermore, the applied monitoring and assessment techniques provide insight into the selection process and can be highly useful to study and improve experimental cell-SELEX designs to increase selection efficiency.

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Phage Display to Augment Biomaterial Function

International Journal of Molecular Sciences ◽

10.3390/ijms21175994 ◽

2020 ◽

Vol 21 (17) ◽

pp. 5994

Author(s):

Thomas A. Davidson ◽

Samantha J. McGoldrick ◽

David H. Kohn

Keyword(s):

Phage Display ◽

High Specificity ◽

Specific Protein ◽

High Affinity ◽

Dual Functional ◽

Cell Tissue ◽

Functional Peptides ◽

Targeting Peptide ◽

Functional Peptide ◽

Protein Cell

Biomaterial design relies on controlling interactions between materials and their biological environments to modulate the functions of proteins, cells, and tissues. Phage display is a powerful tool that can be used to discover peptide sequences with high affinity for a desired target. When incorporated into biomaterial design, peptides identified via phage display can functionalize material surfaces to control the interaction between a biomaterial and its local microenvironment. A targeting peptide has high specificity for a given target, allowing for homing a specific protein, cell, tissue, or other material to a biomaterial. A functional peptide has an affinity for a given protein, cell, or tissue, but also modulates its target’s activity upon binding. Biomaterials can be further enhanced using a combination of targeting and/or functional peptides to create dual-functional peptides for bridging two targets or modulating the behavior of a specific protein or cell. This review will examine current and future applications of phage display for the augmentation of biomaterials.

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Phage-based Electrochemical Sensors: A Review

Micromachines ◽

10.3390/mi10120855 ◽

2019 ◽

Vol 10 (12) ◽

pp. 855 ◽

Cited By ~ 9

Author(s):

Jingting Xu ◽

Ying Chau ◽

Yi-kuen Lee

Keyword(s):

Phage Display ◽

Electrochemical Detection ◽

Electrochemical Sensors ◽

High Specificity ◽

The Other ◽

Detection Techniques ◽

Recognition Element

Phages based electrochemical sensors have received much attention due to their high specificity, sensitivity and simplicity. Phages or bacteriophages provide natural affinity to their host bacteria cells and can serve as the recognition element for electrochemical sensors. It can also act as a tool for bacteria infection and lysis followed by detection of the released cell contents, such as enzymes and ions. In addition, possible detection of the other desired targets, such as antibodies have been demonstrated with phage display techniques. In this paper, the recent development of phage-based electrochemical sensors has been reviewed in terms of the different immobilization protocols and electrochemical detection techniques.

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