{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T02:51:40Z","timestamp":1792723900910,"version":"4.5.0"},"reference-count":64,"publisher":"American Chemical Society (ACS)","issue":"38","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100027043","name":"Milken Institute","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100027043","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Luciferases are widely used bioluminescent reporters, yet access to these enzymes has historically relied on recombinant expression. Using automated fast-flow peptide synthesis (AFPS), we synthesized Gaussia luciferase (GLuc, 18.1 kDa, 168 amino acids), picALuc (13 kDa, 120 amino acids), and LuxSit-i (13.8 kDa, 118 amino acids), representing a nature-derived disulfide-rich luciferase, an engineered luciferase, and a de novo-designed luciferase, respectively. Synthetic GLuc was purified, oxidatively folded, and recovered as a monomeric, enzymatically active enzyme with biophysical properties comparable to its recombinant counterpart. picALuc likewise required oxidative folding, whereas cysteine-free LuxSit-i was folded by denaturant removal using size-exclusion chromatography. Biochemical and biophysical analyses confirmed the structural integrity and activity of the three synthetic enzymes. Notably, LuxSit-i represents, to our knowledge, the original total chemical synthesis of a fully de novo-designed enzyme. Together, these proteins expand the known repertoire of fully synthetic enzymes produced by solid-phase peptide synthesis by approximately 14% and establish the initial examples of fully synthetic luciferases produced by solid-phase peptide synthesis that demonstrate enzymatic activity. These findings position AFPS as a platform for accessing functional enzymes across evolutionary and design spaces and may enable the development of luciferases incorporating noncanonical residues, site-specific modifications, or mirror-image sequences.<\/jats:p>","DOI":"10.1021\/acs.joc.6c01513","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T14:01:46Z","timestamp":1789394506000},"page":"12996-13007","source":"Crossref","is-referenced-by-count":1,"title":["Automated Flow Synthesis\nof Nature-Derived, Engineered,\nand\n                    <i>De Novo<\/i>\n                    -Designed Functional Luciferases"],"prefix":"10.1021","volume":"91","author":[{"given":"David","family":"Sarabia-Castillo","sequence":"first","affiliation":[{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amanda E.","family":"Cowfer Chavez","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunit","family":"Pal","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4919-2296","authenticated-orcid":true,"given":"Charlotte E.","family":"Farquhar","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5640-1645","authenticated-orcid":true,"given":"Andrei","family":"Loas","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7242-801X","authenticated-orcid":true,"given":"Bradley L.","family":"Pentelute","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]},{"name":"Massachusetts Institute of Technology , , 500 Main Street , , ,","place":["Cambridge, Massachusetts, United States, 02142"]},{"name":"Massachusetts Institute of Technology , , 77 Massachusetts Avenue , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,14]]},"reference":[{"issue":"9","key":"2026092509095087200_cit1","doi-asserted-by":"publisher","first-page":"5668","DOI":"10.1039\/D0CS01492C","article-title":"Applications of bioluminescence in biotechnology and\nbeyond","volume":"50","author":"Syed","year":"2021","journal-title":"Chem. 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Physiol."},{"key":"2026092509095087200_cit4","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1186\/1472-6750-8-81","article-title":"Application of the Dual-Luciferase\nReporter Assay to the Analysis of Promoter Activity in Zebrafish Embryos","volume":"8","author":"Alcaraz-Pe\u0301rez","year":"2008","journal-title":"BMC Biotechnol."},{"issue":"5715","key":"2026092509095087200_cit5","doi-asserted-by":"publisher","first-page":"1621","DOI":"10.1126\/science.1105776","article-title":"High-throughput Mapping of a Dynamic Signaling\nNetwork in Mammalian Cells","volume":"307","author":"Barrios-Rodiles","year":"2005","journal-title":"Science"},{"issue":"23","key":"2026092509095087200_cit6","doi-asserted-by":"publisher","first-page":"7870","DOI":"10.1073\/pnas.82.23.7870","article-title":"Cloning of\nfirefly luciferase cDNA and the expression of active luciferase in\nEscherichia coli","volume":"82","author":"de Wet","year":"1985","journal-title":"Proc. Natl. Acad. Sci. U.\nS. 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