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Xenoprotein engineering via synthetic libraries.

ABSTRACT: Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that of cell surface display was achieved by a combination of magnetic bead enrichment, flow cytometry analysis of on-bead screens, and high-throughput MS/MS-based sequencing of identified active compounds. Direct screening of a synthetic protein library by these methods resulted in the de novo discovery of mirror-image miniprotein-based binders to a ?150-kDa protein target, a task that would be difficult or impossible by other means.

SUBMITTER: Gates ZP

PROVIDER: S-EPMC6003312 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Xenoprotein engineering via synthetic libraries.

Gates Zachary P ZP Vinogradov Alexander A AA Quartararo Anthony J AJ Bandyopadhyay Anupam A Choo Zi-Ning ZN Evans Ethan D ED Halloran Kathryn H KH Mijalis Alexander J AJ Mong Surin K SK Simon Mark D MD Standley Eric A EA Styduhar Evan D ED Tasker Sarah Z SZ Touti Faycal F Weber Jessica M JM Wilson Jessica L JL Jamison Timothy F TF Pentelute Bradley L BL

Proceedings of the National Academy of Sciences of the United States of America 20180521 23

Chemical methods have enabled the total synthesis of protein molecules of ever-increasing size and complexity. However, methods to engineer synthetic proteins comprising noncanonical amino acids have not kept pace, even though this capability would be a distinct advantage of the total synthesis approach to protein science. In this work, we report a platform for protein engineering based on the screening of synthetic one-bead one-compound protein libraries. Screening throughput approaching that o ...[more]

PMID: 29784819

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Project description:Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition. Here we describe the generation and experimental validation of two different single-chain antibody variable fragment libraries that efficiently generate binders to peptides, a class of molecules that has proven to be a difficult target for antibody generation. First, a human anti-peptide library was constructed by diversifying a scaffold: the human variable heavy chain (V(H)) germ line gene 3-23, which was fused to a variant of the human variable light chain (V(L)) germ line gene A27, in which L1 was modified to encode the canonical structure found in anti-peptide antibodies. The sequence diversity was introduced into 3-23 (V(H)) only, targeting for diversification residues commonly found in contact with protein and peptide antigens. Second, a murine library was generated using the antibody 26-10, which was initially isolated based on its affinity to the hapten digoxin, but also binds peptides and exhibits a canonical structure pattern typical of anti-peptide antibodies. Diversity was introduced in the V(H) only using the profile of amino acids found at positions that frequently contact peptide antigens. Both libraries yielded binders to two model peptides, angiotensin and neuropeptide Y, following screening by solution phage panning. The mouse library yielded antibodies with affinities below 20 nM to both targets, although only the V(H) had been subjected to diversification.

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Xenoprotein engineering via synthetic libraries.

Publications

Xenoprotein engineering via synthetic libraries.

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