Automated flow synthesis of native, engineered, and de novo-designed functional luciferases
Year: 2026
Type: preprint
Abstract: Luciferases are widely used bioluminescent reporters, yet access to these enzymes has historically relied on recombinant expression. Here, we show that automated fast-flow peptide synthesis (AFPS) enables the rapid production of functional luciferases spanning native, engineered, and de novo-designed scaffolds. Using AFPS, we synthesized Gaussia luciferase (GLuc, 18.1 kDa, 168 amino acids), picALuc (13 kDa, 120 amino acids), and LuxSit-i (13.8 kDa, 120 amino acids), representing a natural 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 LuxSit-i, which lacks cysteine residues, 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 first total chemical synthesis of a fully de novo-designed enzyme. These three luciferases collectively span natural, engineered, and computationally designed enzyme architectures, which represents an approximately 14% expansion of the known repertoire of fully synthetic enzymes produced by solid-phase peptide synthesis. These studies establish the first examples of fully synthetic luciferases produced by solid-phase peptide synthesis that retain native-like enzymatic activity. Together, these findings demonstrate AFPS as a general platform for accessing functional enzymes across evolutionary and design spaces alike. We anticipate that this platform will enable the development of next-generation luciferases through noncanonical residue incorporation, site-specific modification, and mirror-image variants.
Source: ChemRxiv
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7165147033