Citrullination, a type of deimination, is a critical post-translational modification involved in the natural processing of the epidermal protein filaggrin. This modification is catalyzed by peptidyl arginine deiminases (PADs), which convert arginine residues to citrulline. This process facilitates the formation of the skin’s outermost layer and the subsequent degradation of citrullinated filaggrin into natural moisturizing factors. In this study, we characterized PAD-mediated citrullination using the COLDER assay utilizing purified recombinant human PAD1, PAD3, and filaggrin proteins. Citrulline content was quantified based on a distinct colorimetric change, measured spectrophotometrically at 540 nm. Subsequently, the crude extract-based cell-free protein synthesis system was utilized to establish a “one-pot” citrullination reaction. PAD-overexpressing cell extract was combined with filaggrin-encoding DNA in the presence of calcium ions. The reaction was analyzed by SDS-PAGE and western blot, revealing an upward shift in filaggrin molecular weight, indicative of arginine to citrulline conversion. This study not only provides valuable insight into the native citrullination process, but also an opportunity for the biomanufacturing of citrullinated filaggrin in a cost-effective and accessible system.
