title: NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.
abstract: Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M(-1) s(-1), koff = 0.2 s(-1)) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21-37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Dixon
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Marie K
authors.initials: MK
authors.lastname: Schwinn
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Mary P
authors.initials: MP
authors.lastname: Hall
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Kris
authors.initials: K
authors.lastname: Zimmerman
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Otto
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Lubben
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Braeden L
authors.initials: BL
authors.lastname: Butler
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Brock F
authors.initials: BF
authors.lastname: Binkowski
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Machleidt
authors.affiliation: Promega Biosciences Incorporated , San Luis Obispo, California 93401, United States.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kirkland
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Monika G
authors.initials: MG
authors.lastname: Wood
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Eggers
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Lance P
authors.initials: LP
authors.lastname: Encell
authors.affiliation: Promega Corporation , Madison, Wisconsin 53711, United States.
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
mesh_terms: D000595:Amino Acid Sequence; D019390:Arrestins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D049408:Luminescent Agents / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps / Q000187:drug effects; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D049408:Luminescent Agents; D010455:Peptides; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D017868:Cyclic AMP-Dependent Protein Kinases; C094219:beta-lactamase Sme-1; D001618:beta-Lactamases