scalable-high-throughput-selection-from-phage-displayed-synthetic

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

The Recombinant Antibody Network

University of Toronto

Protein interaction networks depend in part on the specific recognition of unstructured peptides by folded domains. Understanding how members of a domain family use a similar fold to recognize different peptide sequences selectively is a fundamental question. One way to advance our understanding of peptide recognition is to apply an existing model of peptide recognition for a particular domain toward engineering synthetic domain variants with desired properties. Successes, failures, and unintended outcomes can help refine the model and can illuminate more general principles of peptide recognition. Using the PDZ domain fold as an example, we describe methods for (1) structure-based combinatorial library design and directed evolution of domain variants and (2) specificity profiling of large repertoires of synthetic variants using multiplexed deep sequencing. Peptide-binding preferences for hundreds of variants can be decoded in parallel, enabling comparisons between different library designs and selection pressures. The tremendous depth of coverage of the binding peptide profiles also permits robust computational analysis. This approach to studying peptide recognition can be applied to other domains and to a variety of structural and functional models by tailoring the combinatorial library design and selection pressures accordingly.

Engineering and analysis of peptide-recognition domain specificities by phage display and deep sequencing.

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.

Alteration of the C-terminal ligand specificity of the erbin PDZ domain by allosteric mutational effects.

Health care providers are seeing an increased number of patients under the influence of several new psychoactive drug classes. Synthetic cannabinoids, cathinones, and piperazines are sought by users for their psychoactive effects, perceived safety profile, minimal legal regulations, and lack of detection on routine urine drug screening. However, these drugs are beginning to be recognized by the medical community for their toxic effects. The neuropsychiatric and cardiovascular toxicities are among the most common reasons for emergency medical treatment, which in some cases, can be severe and even life-threatening. Management strategies are often limited to supportive and symptomatic care due to the limited published data on alternative treatment approaches. The purpose of this article is to offer health care providers, emergency medical personnel in particular, an awareness and understanding of the dangers related to some of the new psychoactive drugs of abuse. The background, pharmacology, toxicity, management, detection, and legal status of each class will be discussed.

Megan E. McLaughlin

The Recombinant Antibody Network

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries