{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:22:55Z","timestamp":1785435775889,"version":"3.56.0"},"posted":{"date-parts":[[2026,2,13]]},"group-title":"Bioengineering","reference-count":48,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T00:00:00Z","timestamp":1770940800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R35GM138265"],"award-info":[{"award-number":["R35GM138265"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"U.S. National Science Foundation","doi-asserted-by":"crossref","award":["2242763"],"award-info":[{"award-number":["2242763"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The rapid engineering of high-affinity binding proteins, such as nanobodies and single-domain antibodies (sdAbs), is increasingly driven by cell-free, machine\u2013learning\u2013guided optimization. However, high-throughput, quantitative characterization of binding affinity remains a major bottleneck, particularly for proteins expressed in cell-free systems without purification. Here, we present High-Throughput FRET Affinity Screening Technique (HTFAST) for rapid affinity characterization of binders expressed directly in crude\n                  <jats:italic>E. coli<\/jats:italic>\n                  cell-free protein synthesis reactions. HTFAST leverages F\u00f6rster resonance energy transfer (FRET) between fluorescent-protein\u2013fused binders and dye-labeled antigens to enable real-time, quantitative measurement of equilibrium dissociation constants. We systematically optimized fluorophore pairs used and labeling parameters using the SpyTag003\u2013SpyCatcher003 model system. Using donor-quenching and acceptor-emission FRET analyses, HTFAST reliably quantified nanomolar binding affinities in crude lysates for SpyTag003\u2013SpyCatcher003 model system. We validated the platform for nanobodies by characterizing a CD4-binding nanobody, Nb457, and benchmarking multiple SARS-CoV-2 receptor-binding domain sdAbs, demonstrating HTFAST\u2019s ability to rank binding strengths across a range of affinities. Finally, we demonstrate that both binding partners can be expressed directly in CFPS, further streamlining screening workflows. Overall, HTFAST provides a scalable, quantitative, and cell-free\u2013compatible approach for high-throughput affinity screening, well suited for DBTL campaigns aimed at accelerating the development of next-generation binding proteins.\n                <\/jats:p>","DOI":"10.64898\/2026.02.12.697512","type":"posted-content","created":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T07:20:13Z","timestamp":1771053613000},"source":"Crossref","is-referenced-by-count":0,"title":["High-Throughput FRET Affinity Screening Technique (HTFAST) For Cell-Free Expressed Binding Protein Characterization"],"prefix":"10.64898","author":[{"given":"Sepehr","family":"Hejazi","sequence":"first","affiliation":[{"name":"Chemical and Biological Engineering \u2013 Iowa State University, 618 Bissell Rd, Ames, IA 50011"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimia","family":"Noroozi","sequence":"additional","affiliation":[{"name":"Chemical and Biological Engineering \u2013 Iowa State University, 618 Bissell Rd, Ames, IA 50011"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vito","family":"Jurasic","sequence":"additional","affiliation":[{"name":"Chemical and Biological Engineering \u2013 Iowa State University, 618 Bissell Rd, Ames, IA 50011"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura R","family":"Jarboe","sequence":"additional","affiliation":[{"name":"Chemical and Biological Engineering \u2013 Iowa State University, 618 Bissell Rd, Ames, IA 50011"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nigel F","family":"Reuel","sequence":"additional","affiliation":[{"name":"Chemical and Biological Engineering \u2013 Iowa State University, 618 Bissell Rd, Ames, IA 50011"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026021607301001000_2026.02.12.697512v1.1","doi-asserted-by":"publisher","DOI":"10.1186\/s12929-019-0592-z"},{"key":"2026021607301001000_2026.02.12.697512v1.2","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1186\/s12951-025-03169-5","article-title":"Unveiling the new chapter in nanobody engineering: advances in traditional construction and AI-driven optimization","volume":"23","year":"2025","journal-title":"J Nanobiotechnol"},{"key":"2026021607301001000_2026.02.12.697512v1.3","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-007-1142-2"},{"key":"2026021607301001000_2026.02.12.697512v1.4","doi-asserted-by":"publisher","DOI":"10.1007\/s40259-019-00392-z"},{"key":"2026021607301001000_2026.02.12.697512v1.5","doi-asserted-by":"crossref","first-page":"6961","DOI":"10.1038\/s41467-024-51414-6","article-title":"Highly potent and broadly neutralizing anti-CD4 trimeric nanobodies inhibit HIV-1 infection by inducing CD4 conformational alteration","volume":"15","year":"2024","journal-title":"Nat Commun"},{"key":"2026021607301001000_2026.02.12.697512v1.6","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/S0167-4838(99)00030-8","article-title":"Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies","volume":"1431","year":"1999","journal-title":"Biochimica et Biophysica Acta (BBA) - 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