Development of venus-fused Cypridina luciferase (VCL) and specific nanobody-tethering assay to model kleptoprotein uptake.

The bioluminescent fish Parapriacanthus ransonneti does not synthesize the luciferase enzyme required for light emission; instead, it acquires the high-molecular-weight enzyme from its prey, the ostracod Cypridina noctiluca. This unique phenomenon, termed "kleptoprotein", represents a novel mode of phenotype acquisition where exogenous biopolymers are functionally sequestered. Unlike complex organelle sequestration (e.g., kleptoplasty), the kleptoprotein system involves a single protein, offering a tractable model to elucidate the molecular mechanisms of exogenous protein uptake. However, the mechanisms underlying luciferase internalization remain unknown due to the lack of tools to visualize and quantify this process. Here, we developed a dual-function probe by fusing the fluorescent protein Venus to C. noctiluca luciferase (VCL). Guided by AlphaFold 3 structural simulations predicting stable binding and catalytic integrity, we engineered a membrane-tethered Venus-trap system to validate VCL as a tracer. We demonstrate that VCL specifically interacts with the surface-displayed nanobody. Fluorescence imaging visualized VCL binding at trap-plasmid concentrations ≥ 50 ng/mL and VCL concentrations ≥ 2000 ng/mL. The bioluminescence assay exhibited superior sensitivity, detecting binding at VCL concentrations as low as 200 ng/mL. These results confirm that the fusion protein remains functionally intact and offers a highly sensitive detection method. This VCL-trap system establishes a robust platform for identifying the putative luciferase receptor and dissecting the intracellular dynamics of kleptoprotein bioluminescence.