Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor.

Abstract

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6×His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd = 1.4 ± 0.4 nM, n = 3), the overexpressed LIFR/gp130 (Kd = 115 ± 8 pM, n = 3), and the endogenously expressed LIFR/gp130 (Kd = 33.1 ± 3.2 pM, n = 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligand-receptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.