In Vitro Analysis of Profilin 1 Binding to Recombinant Actin.

Profilin 1 (PFN1) is a key actin-binding protein (ABP) involved in cytoskeletal dynamics, regulating actin polymerization and filament remodeling. While native actin has been extensively studied, recombinant actin produced through biotechnological approaches remains less explored due to solubility and folding challenges. Previously, we assessed the effects of S-nitrosylation on actin binding, providing insights into post-translational modifications that regulate actin function. In the present study, we provide a comprehensive protocol to produce recombinant β-actin using an in vitro transcription-translation system supplemented with chaperonin CCT to analyze its interaction with PFN1. Our findings highlight the utility of recombinant actin for studying ABPs and their regulatory mechanisms, offering a cost-effective alternative for structural and functional analyses.