Retooling phage display with electrohydrodynamic nanomixing and nanopore sequencing

Lab on a Chip ◽  
2019 ◽  
Vol 19 (24) ◽  
pp. 4083-4092 ◽  
Author(s):  
Lyndon J. Raftery ◽  
Christopher B. Howard ◽  
Yadveer S. Grewal ◽  
Ramanathan Vaidyanathan ◽  
Martina L. Jones ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Nanopore Sequencing ◽  
Phage Display Libraries ◽  
Target Binding

High throughput screening of phage display libraries for target binding molecules using electrohydrodynamic nanomixing and nanopore sequencing.

scholarly journals Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

2005 ◽  
Vol 10 (2) ◽  
pp. 108-117 ◽  
Author(s):  
Thomas Vanhercke ◽  
Christophe Ampe ◽  
Luc Tirry ◽  
Peter Denolf
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Enrichment ◽  
Phage Display Libraries ◽  
Viral Particles ◽  
Phage Libraries ◽  
Rapid Characterization

Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phage-displayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 104-fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries.

scholarly journals Creation of Novel Protein Transduction Domain (PTD) Mutants by a Phage Display-Based High-Throughput Screening System

2006 ◽  
Vol 29 (8) ◽  
pp. 1570-1574 ◽  
Author(s):  
Yohei Mukai ◽  
Toshiki Sugita ◽  
Tomoko Yamato ◽  
Natsue Yamanada ◽  
Hiroko Shibata ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Screening System ◽  
Novel Protein

scholarly journals Directed Evolution of Aptamer Discovery Technologies

2021 ◽  
Author(s):  
Diana Wu ◽  
Chelsea Gordon ◽  
John Shin ◽  
Michael Eisenstein ◽  
Hyongsok Tom Soh
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Click Reaction ◽  
Clinical Diagnostics ◽  
High Quality ◽  
Affinity Reagents ◽  
Wide Range ◽  
Target Binding ◽  
Modified Nucleobases ◽  
Parallel Fashion

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components

2018 ◽  
Vol 456 ◽  
pp. 44-53 ◽  
Author(s):  
Pablo San Segundo-Acosta ◽  
María Garranzo-Asensio ◽  
Carmen Oeo-Santos ◽  
Ana Montero-Calle ◽  
Joaquín Quiralte ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Methodological Approach ◽  
Protein Microarrays ◽  
T7 Phage Display ◽  
T7 Phage

scholarly journals High-Throughput Method for Ranking the Affinity of Peptide Ligands Selected from Phage Display Libraries

2008 ◽  
Vol 19 (5) ◽  
pp. 993-1000 ◽  
Author(s):  
A. González-Techera ◽  
M. Umpiérrez-Failache ◽  
S. Cardozo ◽  
G. Obal ◽  
O. Pritsch ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
Peptide Ligands ◽  
Phage Display Libraries ◽  
High Throughput Method

Phage Display of Functional Human TNF-α Converting Enzyme Catalytic Domain: A Rapid Method for the Production of Stabilized Proteolytic Proteins for Assay Development and High-Throughput Screening

2002 ◽  
Vol 7 (5) ◽  
pp. 433-440 ◽  
Author(s):  
Yangde Chen ◽  
Katrina Diener ◽  
Indravadan R. Patel ◽  
John K. Kawooya ◽  
Gary A. Martin ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Catalytic Domain ◽  
Assay Development ◽  
Scale Production ◽  
Converting Enzyme ◽  
Display System ◽  
Tnf Α

The catalytic domain of human tumor necrosis factor-α (TNF-α) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-α to generate the mature TNF-α in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-α-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 °C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 °C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

scholarly journals One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Emanuele Sasso ◽  
Rolando Paciello ◽  
Francesco D’Auria ◽  
Gennaro Riccio ◽  
Guendalina Froechlich ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
Phage Display ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Hepatitis C Virus Infection ◽  
Novel Approach ◽  
Phage Display Libraries ◽  
Junction Protein

Expanding the availability of monoclonal antibodies interfering with hepatitis C virus infection of hepatocytes is an active field of investigation within medical biotechnologies, to prevent graft reinfection in patients subjected to liver transplantation and to overcome resistances elicited by novel antiviral drugs. In this paper, we describe a complete pipeline for screening of phage display libraries of human scFvs against native Claudin-1, a tight-junction protein involved in hepatitis C virus infection, expressed on the cell surface of human hepatocytes. To this aim, we implemented a high-throughput sequencing approach for library screening, followed by a simple and effective strategy to recover active binder clones from enriched sublibraries. The recovered clones were successfully converted to active immunoglobulins, thus demonstrating the effectiveness of the whole procedure. This novel approach can guarantee rapid and cheap isolation of antibodies for virtually any native antigen involved in human diseases, for therapeutic and/or diagnostic applications.

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