Antibody homotypic interactions are encoded by germline light chain complementarity determining region 2.

The utilization of avidity to drive and tune functional responses is fundamental to antibody biology and often underlies the mechanisms of action of monoclonal antibody drugs. There is increasing evidence that antibodies leverage homotypic interactions to enhance avidity, often through weak transient interfaces whereby self-association is coupled with target binding. Here, we comprehensively map the Fab–Fab interfaces of antibodies targeting DR5 and 4-1BB that utilize homotypic interaction to promote receptor activation and demonstrate that both antibodies have similar self-association determinants primarily encoded within a germline light chain complementarity determining region 2 (CDRL2).

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture.

CellCelector™ as a platform in isolating primary B cells for antibody discovery.

The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery.

Multivalent designed proteins protect against SARS-CoV-2 variants of concern.

Unlabelled: Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding.

Correction: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin.

[This corrects the article DOI: 10.1371/journal.pone.

Heat-enhanced peptide synthesis on Teflon-patterned paper.

In this report, we describe the methodology for 96 parallel organic syntheses of peptides on Teflon-patterned paper assisted by heating with an infra-red lamp. SPOT synthesis is an important technology for production of peptide arrays on a paper-based support for rapid identification of peptide ligands, epitope mapping, and identification of bio-conjugation reactions. The major drawback of the SPOT synthesis methodology published to-date is suboptimal reaction conversion due to mass transport limitations in the unmixed reaction spot.

Next-generation sequencing of phage-displayed peptide libraries.

Genetically encoded peptide libraries enabled the discovery of ligands for clinically relevant targets and functional materials. Next-generation sequencing (NGS) of these libraries improved the selection of ligands by detecting low abundant clones and quantifying changes in copy numbers of clones without many rounds of selection. Although NGS platforms have been widely used in genome assembly, quantification of gene expression (RNA-seq), and metagenomic analyses, few examples in the literature describe sequencing phage libraries.

Flow-through synthesis on Teflon-patterned paper to produce peptide arrays for cell-based assays.

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell-based assays. Solvent-repelling barriers made of Teflon-impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow-through syntheses on paper. The confinement and flow-through mixing significantly improves the peptide yield and simplifies the automation of this synthesis.

Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin.

Error analysis of deep sequencing of phage libraries: peptides censored in sequencing.

Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries.

Prospective identification of parasitic sequences in phage display screens.

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.

Discovery of light-responsive ligands through screening of a light-responsive genetically encoded library.

Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library.

Deep sequencing analysis of phage libraries using Illumina platform.

This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target.

Uniform amplification of phage display libraries in monodisperse emulsions.

In this paper, we describe a complete experimental setup for the uniform amplification of libraries of phage. Uniform amplification, which multiplies every phage clone by the same amount irrespective of the growth rate of the clone is essential for phage-display screening. Amplification of phage libraries in a common solution is often non-uniform: it favors fast-growing clones and eliminates those that grow slower.

Quantitative synthesis of genetically encoded glycopeptide libraries displayed on M13 phage.

Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.

Triterpenoidal alkaloids from Buxus natalensis and their acetylcholinesterase inhibitory activity.

Acetylcholinesterase (AChE) inhibition-directed phytochemical studies on the methanolic extract of Buxus natalensis, collected in South Africa, resulted in the isolation of 12 compounds: O(2)-natafuranamine (1), O(10)-natafuranamine (2), cyclonataminol (3), 31-demethylbuxaminol A (4), buxaminol A (5), buxafuranamide (6), buxalongifolamidine (7), buxamine A (8), cyclobuxophylline K (9), buxaminol C (10), methyl syringate (11), and p-coumaroylputrescine (12). Compounds 1-4 were new alkaloids, and compound 5 was isolated for the first time as a natural product. Their structures were elucidated with the aid of extensive NMR and mass spectroscopic studies.