Quick preparation of nanoluciferase-based tracers for novel bioluminescent receptor-binding assays of protein hormones: Using erythropoietin as a model.
Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the so far brightest bioluminescence. In recent studies, we developed NanoLuc as an ultrasensitive probe for novel bioluminescent receptor-binding assays of some protein/peptide hormones. In the present study, we proposed a simple method for quick preparation of the NanoLuc-based protein tracers using erythropoietin (Epo) as a model. Epo is a glycosylated cytokine that promotes erythropoiesis by binding and activating the cell membrane receptor EpoR. For quick preparation of a bioluminescent Epo tracer, an Epo-Luc fusion protein carrying a NanoLuc-6 × His-tag at the C-terminus was secretorily overexpressed in transiently transfected human embryonic kidney (HEK) 293 T cells. The Epo-Luc fusion protein retained high-binding affinities with EpoR either overexpressed in HEK293T cells or endogenously expressed in mouse erythroleukemia cells, representing a novel ultrasensitive bioluminescent tracer for non-radioactive receptor-binding assays. Sufficient Epo-Luc tracer for thousands of assays could be quickly obtained within 2 days through simple transient transfection. Thus, our present work provided a simple method for quick preparation of novel NanoLuc-based bioluminescent tracers for Epo and some other protein hormones to facilitate their ligand-receptor interaction studies.
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Ge
G
Song
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Qing-Ping
QP
Wu
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Ting
T
Xu
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Ya-Li
YL
Liu
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
Zeng-Guang
ZG
Xu
Proteomic and Molecular Enzymology Lab, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
Shi-Fu
SF
Zhang
Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China. Electronic address: zhan-yun.guo@tongji.edu.cn.
Zhan-Yun
ZY
Guo
D000595:Amino Acid Sequence; D000818:Animals; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D008969:Molecular Sequence Data; D049329:Nanostructures / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D008156:Luciferases
Bioluminescence; Epo; NanoLuc; Receptor-binding; Tracer