Xenoprotein engineering via synthetic libraries
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 appr...
| Main Authors: | , , , , , , , , , , , , , , , , , |
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| Format: | Article |
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National Academy of Sciences (U.S.)
2019
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| Online Access: | http://hdl.handle.net/1721.1/120509 https://orcid.org/0000-0003-0588-6157 https://orcid.org/0000-0002-5508-0963 https://orcid.org/0000-0003-4995-1424 https://orcid.org/0000-0002-9383-2185 https://orcid.org/0000-0001-6226-8625 https://orcid.org/0000-0001-5474-4273 https://orcid.org/0000-0003-1632-5195 https://orcid.org/0000-0002-6214-7513 https://orcid.org/0000-0003-2315-6983 https://orcid.org/0000-0001-9021-0308 https://orcid.org/0000-0002-8434-0066 https://orcid.org/0000-0002-8601-7799 |
| Summary: | 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. Keywords: xenoprotein; mirror-image miniprotein; D-protein; protein engineering; flow cytometry |
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