Next-Generation Phage Display to Identify Peptide Ligands of Deubiquitinases.

Phage display (PD) is a powerful method and has been extensively used to generate monoclonal antibodies and identify epitopes, mimotopes, and protein interactions. More recently, the combination of next-generation sequencing (NGS) with PD (NGPD) has revolutionized the capabilities of the method by creating large data sets of sequences from affinity selection-based approaches (biopanning) otherwise challenging to obtain. NGPD can monitor motif enrichment, allow tracking of the selection process over consecutive rounds, and highlight unspecific binders. To tackle the wealth of data obtained, bioinformatics tools have been developed that allow for identifying specific binding sequences (binders) that can then be validated. Here, we provide a detailed account of the use of NGPD experiments to identify ubiquitin-specific protease peptide ligands.

A Simple Whole-Plasmid PCR Method to Construct High-Diversity Synthetic Phage Display Libraries.

Phage display technology utilises peptide and antibody libraries with very high diversities to select ligands with specific binding properties. The production of such libraries can be labour intensive and technically challenging and whilst there are commercial sources of libraries, the exploitation of the resulting binders is constrained by ownership of the libraries. Here, a peptide library of ~ 1 × 109 variants for display on gene VIII was produced alongside three VHH antibody libraries with similar diversity, where 12mer, 16mer or 21mer CDR3s were introduced into the highly stable cAbBCII10 scaffold displayed on gene III. The cloning strategy used a simple whole-plasmid PCR method and type IIS restriction enzyme assembly that facilitate the seamless insertion of diversity into any suitable phage coat protein or antibody scaffold. This method reproducibly produced 1 × 109 variants from just 10 transformations and the four libraries had relatively low bias with 82 to 86% of all sequences present as single copies. The functionality of both peptide and antibody libraries were demonstrated by selection of ligands with specific binding properties by biopanning. The peptide library was used to epitope map a monoclonal antibody. The VHH libraries were pooled and used to select an antibody to recombinant human collagen type 1.

Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

Discovery of peptide ligands targeting a specific ubiquitin-like domain-binding site in the deubiquitinase USP11.

Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 µm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro Furthermore, a crystal structure of a USP11-peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket-deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination-mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals.

Serological surveillance and vaccination are important strategies for controlling infectious diseases of food production animals. However, the compatibility of these strategies is limited by a lack of assays capable of differentiating infected from vaccinated animals (DIVA tests) for established killed or attenuated vaccines. Here, we used next generation phage-display (NGPD) and a 2-proportion Z score analysis to identify peptides that were preferentially bound by IgY from chickens infected with Salmonella Typhimurium or S. Enteritidis compared to IgY from vaccinates, for both an attenuated and an inactivated commercial vaccine. Peptides that were highly enriched against IgY from at least 4 out of 10 infected chickens were selected: 18 and 12 peptides for the killed and attenuated vaccines, respectively. The ten most discriminatory peptides for each vaccine were identified in an ELISA using a training set of IgY samples. These peptides were then used in multi-peptide assays that, when analysing a wider set of samples from infected and vaccinated animals, diagnosed infection with 100% sensitivity and specificity. The data describes a method for the development of DIVA assays for conventional attenuated and killed vaccines.

Mapping polyclonal antibody responses to bacterial infection using next generation phage display.

Mapping polyclonal antibody responses to infectious diseases to identify individual epitopes has the potential to underpin the development of novel serological assays and vaccines. Here, phage-peptide library panning coupled with screening using next generation sequencing was used to map antibody responses to bacterial infections. In the first instance, pigs experimentally infected with Salmonella enterica serovar Typhimurium was investigated. IgG samples from twelve infected pigs were probed in parallel and phage binding compared to that with equivalent IgG from pre-infected animals. Seventy-seven peptide mimotopes were enriched specifically against sera from multiple infected animals. Twenty-seven of these peptides were tested in ELISA and twenty-two were highly discriminatory for sera taken from pigs post-infection (P < 0.05) indicating that these peptides are mimicking epitopes from the bacteria. In order to further test this methodology, it was applied to differentiate antibody responses in poultry to infections with distinct serovars of Salmonella enterica. Twenty-seven peptides were identified as being enriched specifically against IgY from multiple animals infected with S. Enteritidis compared to those infected with S. Hadar. Nine of fifteen peptides tested in ELISA were highly discriminatory for IgY following S. Enteritidis infection (p < 0.05) compared to infections with S. Hadar or S. Typhimurium.

Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets.

Recently the analytical power of the latest high throughput next generation DNA sequencing platforms has been used to analyse phage that have been selected from the panning of large combinatorial libraries displaying either peptide or antibody ligands. This process, commonly referred to as next generation phage display (NGPD), allows the researcher to determine the identity of specific phage that are being enriched against an antigen target by analysis of the DNA sequence encoding the displayed ligand. This method bypasses several steps in conventional phage panning that include laborious colony picking and functional ligand screening. A downside of this approach is that the only output from such experiments is the DNA sequence information of such enriched phage particles. In the case of peptides, the peptide sequence can be synthesised directly and used for further screening; however this is more difficult with larger antibody fragments such as ScFvs. In the case of ScFvs, their coding sequence would have to be fully elucidated, synthesised and re-cloned before expression. We describe here the application of an inverse PCR-ligation methodology that enables the specific recovery of ScFvs of interest from enriched sub-libraries of phage clones. Phagemid particles are recovered using sequence information derived from their unique heavy chain CDR3/FR4 domains and specific clones can be recovered irrespective of CDR3 size and at levels of abundance that would be refractory to their discovery during conventional phage panning and screening.

Expression of a cDNA encoding a member of the hexamerin storage proteins from the moth Sesamia nonagrioides (Lef.) during diapause.

We isolated and sequenced a cDNA clone corresponding to a storage protein (SnoSP1) from the corn stalk borer Sesamia nonagrioides (Lef.). The cDNA for SnoSP1 (2403 bp) codes for a 751 residue protein with predicted molecular mass of 88.3 kDa and calculated isoelectric point pI=8.72. A signal peptide of 16 amino acids is present at the N-terminus and the protein contained conserved insect larval storage protein signature sequence patterns. Multiple alignment analysis of the amino acid sequence revealed that SnoSP1 is most similar to the basic juvenile hormone-suppressible protein 2 precursor (TniSP2) from Trichoplusia ni (71% identity) and other moderately methionine-rich hexamers. According to both phylogenetic analyses and the criteria of amino acid composition, SnoSP1 belongs to the subfamily of moderately methionine-rich storage proteins (3.7% methionine, 11% aromatic amino acid). Treatment with the juvenile hormone analog, methroprene, after head ligation of larvae, is found to suppress the level of SnoSP1 gene, indicating hormonal effects at the transcriptional level. We also examined developmental profiles of SnoSP1 expression in fat body from diapausing and non-diapausing larvae by semi-quantitative and Real-Time PCR assays. In non diapause conditions the abundance of SnoSP1 was found in high levels during the last larval stage and decreased gradually during the pupal stage. Very low levels of this mRNA were detected in larvae that were preparing to enter diapause, but mRNA dramatically increased in those that were in diapause as well as in those that terminate diapause.