Curated Optogenetic Publication Database

Abstract: Fluorescent molecules are essential for bioimaging and visualizing cellular localization, functionalities, including biosensing, ion sensing, and photochromism. The photocleavable fluorescent protein PhoCl1 belongs to a sub-class of green-to-red photoconvertible β-barrel fluorescent protein and has a characteristic green fluorescence conferred by the chromophore p-HBI. In contrast to other photoconvertible proteins, that shift their fluorescence from green-to-red upon photoexposure, PhoCl1 has been reported to render itself non-fluorescent by releasing the 9 amino-acid C-terminal peptide fragment (CTPF) bearing the photo-transformed red chromophore from the β-barrel. Here we show the fate of photoreleased chromophore which shows an unexpected dim red fluorescence. We attribute this dim red fluorescence to the aggregation of CTPF molecules which is validated through dynamic light scattering measurements. We further characterize the aggregated CTPF through various optical techniques to determine the excitation/emission maxima, fluorescence lifetime, quantum yield and rotational correlation time through fluorescence anisotropy. We assessed the red fluorescence behavior under diverse environmental conditions including variations in pH, NaCl, and temperature. Molecular dynamics simulations support our experimentally observed aggregation of CTPF molecules. We supplemented these studies with quantum mechanics/molecular mechanics study which indicated the role of the chromophore in the photodissociated peptide fragment in the generation of dim red fluorescence. These findings not only provide insight into the behavior of fluorescent chromophore-peptide conjugate but also potentially lay the groundwork for developing light-activated fluorescence systems, AIE-based biosensors, and tunable biomaterials for protein tagging and responsive material design.

Abstract: The production of recombinant proteins has become a focal point in biotechnology, with potential applications in catalysis, therapeutics, and diagnostics. Before their application, these proteins undergo cumbersome downstream processing, including multiple resin-based chromatography steps (ion exchange or affinity-based) to isolate the protein of interest from host cell proteins, which are more abundant. These methods often involve (1) nonspecific binding of host cell proteins onto the resin, (2) a trial and error approach in determining elution conditions for the protein of interest, and (3) complex functionalization of the resin. These processes are also further supplemented with additional processing steps including buffer exchange through dialysis or desalting. Despite the prevalence and need for proteins, challenges persist in optimizing elution conditions and minimizing downstream processing steps, which contribute to the overall cost, impeding their translation into the market. To address these challenges, there has been a growing interest in stimuli-responsive purification systems, which allow for precise control and modulation of the purification process for protein recovery by altering their properties or behavior in response to specific external conditions, such as heat, light, or chemicals. We have developed a light-activated molecular purification (LAMP) system, a stimuli-responsive chromatography technique where the purification of recombinant proteins is triggered by light. We employed a photocleavable protein (PhoCl1) that binds specifically to Ni-NTA resin through a hexa-histidine tag at its N-terminus. We harnessed the ability of PhoCl1 to undergo photocleavage into two fragments for the development of LAMP. To demonstrate LAMP, the protein of interest (POI) is genetically fused to the C-terminus of PhoCl1. The exposure to 405 nm light (1.5 mW cm-2 for 12 h) results in the release of POI into the supernatant. We showcased the potential of LAMP by purifying highly charged green fluorescent proteins and an enzyme, riboflavin kinase. Our custom-built violet light LED setup achieved more than 50% light-induced photocleavage of the fusion constructs, resulting in the release of more than 30% of the POI into the supernatant, with the remainder retained within the resin. All the proteins purified using LAMP were more than 90% pure. Moreover, the comparison of the riboflavin kinase purified through LAMP and the traditional chromatography (Ni-NTA affinity method) revealed no significant changes in the activity levels. These highlight the broad potential of LAMP in providing a facile, yet robust stimuli-responsive protein purification technique, which leverages the potential of light to purify the proteins and overcome the limitations of current conventional chromatography systems.