Light‐Activated Molecular Purification (LAMP): A Photo‐Responsive Affinity‐Chromatography Protocol for Recombinant Protein Purification

Recombinant protein purification is a critical step prior to their use in therapeutics and diagnostics. Affinity and ion-exchange chromatography are among the most widely used purification strategies; however, both rely on extensive trial-and-error optimization and require chemical additives or high salt concentrations for protein elution. These conditions necessitate additional, time-consuming downstream dialysis steps to remove residual chemicals. To address these limitations, we propose a light-triggered protein elution strategy that eliminates the need for chemical elution agents and simplifies downstream processing. This approach exploits PhoCl1, a photocleavable protein that undergoes light-induced cleavage into an N-terminal empty barrel and a short C-terminal peptide fragment (Zhang et al., 2017; Lu et al., 2021). We engineered a fusion construct in which a 6×His tag is positioned at the N-terminus of PhoCl1, while the protein of interest (POI) is fused to its C-terminus (6×His-PhoCl1-POI). Following immobilization of the whole-cell lysate on Ni-NTA resin and removal of nonspecifically bound proteins, light exposure induces PhoCl1 cleavage. The His-tagged N-terminal fragment remains bound to the resin, while the POI is selectively released into the supernatant. Using this method, we have achieved protein purities exceeding 90%, demonstrating a rapid, reagent-free, and highly efficient alternative to conventional protein purification workflows. Here, for demonstration purposes, we have included a POI, riboflavin kinase (MjRibK) found in Methanocaldococcus jannaschii. In this protocol, we describe the design, expression, and characterization of PhoCl1 fusion proteins using an intracellular expression system in Escherichia coli, with all steps optimized for cytosolic production. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Light-Activated Affinity-Based Recombinant Protein Purification.