Light-Activated\nMolecular Purification (LAMP) of Recombinant\nProteins

The production of recombinant proteins has become a focal\npoint\nin biotechnology, with potential applications in catalysis, therapeutics,\nand diagnostics. Before their application, these proteins undergo\ncumbersome downstream processing, including multiple resin-based chromatography\nsteps (ion exchange or affinity-based) to isolate the protein of interest\nfrom host cell proteins, which are more abundant. These methods often\ninvolve (1) nonspecific binding of host cell proteins onto the resin,\n(2) a trial and error approach in determining elution conditions for\nthe protein of interest, and (3) complex functionalization of the\nresin. These processes are also further supplemented with additional\nprocessing steps including buffer exchange through dialysis or desalting.\nDespite the prevalence and need for proteins, challenges persist in\noptimizing elution conditions and minimizing downstream processing\nsteps, which contribute to the overall cost, impeding their translation\ninto the market. To address these challenges, there has been a growing\ninterest in stimuli-responsive purification systems, which allow for\nprecise control and modulation of the purification process for protein\nrecovery by altering their properties or behavior in response to specific\nexternal conditions, such as heat, light, or chemicals. We have developed\na light-activated molecular purification (LAMP) system, a stimuli-responsive\nchromatography technique where the purification of recombinant proteins\nis triggered by light. We employed a photocleavable protein (PhoCl1)\nthat binds specifically to Ni-NTA resin through a hexa-histidine tag\nat its N-terminus. We harnessed the ability of PhoCl1 to undergo photocleavage\ninto two fragments for the development of LAMP. To demonstrate LAMP,\nthe protein of interest (POI) is genetically fused to the C-terminus\nof PhoCl1. The exposure to 405 nm light (1.5 mW cm<sup>–2</sup> for 12 h) results in the release of POI into the supernatant. We\nshowcased the potential of LAMP by purifying highly charged green\nfluorescent proteins and an enzyme, riboflavin kinase. Our custom-built\nviolet light LED setup achieved more than 50% light-induced photocleavage\nof the fusion constructs, resulting in the release of more than 30%\nof the POI into the supernatant, with the remainder retained within\nthe resin. All the proteins purified using LAMP were more than 90%\npure. Moreover, the comparison of the riboflavin kinase purified through\nLAMP and the traditional chromatography (Ni-NTA affinity method) revealed\nno significant changes in the activity levels. These highlight the\nbroad potential of LAMP in providing a facile, yet robust stimuli-responsive\nprotein purification technique, which leverages the potential of light\nto purify the proteins and overcome the limitations of current conventional\nchromatography systems.