title: 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.
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China; Shanghai Toneker Biotechnology Co. Ltd., Shanghai 200092, China.
authors.forename: Sheng-Xiang
authors.initials: SX
authors.lastname: He
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
authors.forename: Ge
authors.initials: G
authors.lastname: Song
authors.affiliation: Shanghai Toneker Biotechnology Co. Ltd., Shanghai 200092, China.
authors.forename: Jia-Ping
authors.initials: JP
authors.lastname: Shi
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Guo
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China. Electronic address: zhan-yun.guo@tongji.edu.cn.
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D050822:Cytokine Receptor gp130 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000235:genetics / Q000378:metabolism*; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism
chemical_list: C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D050796:STAT3 Transcription Factor; D050822:Cytokine Receptor gp130; D008156:Luciferases
keywords: Bioluminescence; Leukemia inhibitory factor; Nanoluciferase; Protein fusion tag; Receptor-binding assays