Abstract

Light-Oxygen-Voltage (LOV) domains are ubiquitous photosensory modules, often employed in optogenetic tools, whose function relies on the reversible formation of a covalent bond between a conserved cysteine residue and a flavin mononucleotide (FMN) cofactor. This study addresses the challenge of producing a stable, non-photoreactive LOV domain from an archaeal source, specifically engineered to be cysteine-less, which is critical for applications requiring a stable, non-covalent FMN-binding scaffold. The primary objective was the high-yield expression and purification of this novel **archaeal cysteine-less LOV domain** variant. Using a standard *E. coli* expression system, the protein was successfully produced and purified to homogeneity via a two-step chromatographic process, yielding milligram quantities of highly pure protein. Spectroscopic analysis confirmed the stable, non-covalent binding of the FMN cofactor, demonstrating that the cysteine mutation successfully abrogates the characteristic photoreactivity while maintaining the structural integrity of the binding pocket. This purified, stable LOV domain variant serves as an invaluable biochemical tool for structural studies, particularly in understanding the fundamental mechanisms of FMN-protein interaction without the confounding variable of photochemistry. Furthermore, it represents a promising, non-photosensitive scaffold for the development of novel biosensors and protein-based reagents in cell biology and biochemistry.