Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique.

Irina A. Ishina,Leyla A. Ovchinnikova,Alexey A. Belogurov,Ioanna N. Filimonova,Yakov A. Lomakin,Maria Y. Zakharova,Azad E. Mamedov

doi:10.3390/ijms21176258

Abstract

Effective and versatile screening of the peptide ligands capable of selectively binding to diverse receptors is in high demand for the state-of-the-art technologies in life sciences, including probing of specificity of the cell surface receptors and drug development. Complex microenvironment and structure of the surface receptors significantly reduce the possibility to determine their specificity, especially when in vitro conditions are utilized. Previously, we designed a publicly available platform for the ultra-high-throughput screening (uHTS) of the specificity of surface-exposed receptors of the living eukaryotic cells, which was done by consolidating the phage display and flow cytometry techniques. Here, we significantly improved this methodology and designed the fADL-1e-based phage vectors that do not require a helper hyperphage for the virion assembly. The enhanced screening procedure was tested on soluble human leukocyte antigen (HLA) class II molecules and transgenic antigen-specific B cells that express recombinant lymphoid B-cell receptor (BCR). Our data suggest that the improved vector system may be successfully used for the comprehensive search of the receptor ligands in either cell-based or surface-immobilized assays.

Highlights

Despite the remarkable advances made by molecular biology techniques, determining the specificity of the antigen receptors is still a non-trivial and challenging task
In the present report we demonstrated that combining the phage display technique and fluorescence-activated cell sorting (FACS) can dramatically improve the screening of the peptide ligands receptors with an unknown or complex structure in their natural environment
CyCLOPS platform may be effectively used for elucidation of the etiology of the autoimmune diseases, in which there is no clear understanding what autoantigens are involved in the disease triggering

Summary

Introduction

Despite the remarkable advances made by molecular biology techniques, determining the specificity of the antigen receptors is still a non-trivial and challenging task. Various screening approaches to investigating the antigen-specific cells have been undertaken in the last few years. The 10 × Genomics platform was used by Goldstein and colleagues for the high-throughput sequencing of the antigen-specific individual B-cell receptors (BCR), located directly on the living B lymphocytes, that recognize the desired protein [4]. LIBRA-seq developed by Setliff et al allows identifying the BCRs for several barcoded antigens simultaneously [5]. The main limitation of the described techniques is the restricted number of the antigens that can be analyzed in a single experiment, as each individual protein should be expressed, purified, and labeled separately. A library of the bacteriophages that would simultaneously carry up to millions of the DNA-encoded ligands seems to be a beneficial alternative to the barcoded recombinant proteins

Objectives

Methods

Results

Conclusion