Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins

Abstract

Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies on genetic fusion of luciferases to antibodies or nonspecific chemical conjugation strategies. Here, we report a generic method to generate well-defined covalent antibody− luciferase conjugates starting from commercially available monoclonal antibodies. Our approach uses fusion proteins consisting of the bright blue light-emitting luciferase NanoLuc (NL) and an Fcbinding protein domain (Gx) that can be photo-cross-linked to the antibody using UV light illumination. Green and red color variants were constructed by tight fusion of the NanoLuc with a green fluorescent acceptor domain and introduction of Cy3, respectively. To increase the already bright NanoLuc emission, tandem fusions were successfully developed in which the Gx domain is fused to two or three copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photo-cross-linked to all human immunoglobulin G (IgG) isotypes and most mammalian IgG's using 365 nm light, yielding antibodies with either one or two luciferase domains. The bioluminescent antibodies were successfully used in cell immunostaining and bioanalytical assays such as enzyme-linked immunosorbent assay (ELISA) and Western blotting.

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…To overcome the problem, several original approaches were developed. So, the bioorthogonal conjugation or photoconjugation methods of obtaining luciferase-antibody conjugates that would provide a high conjugate yield, control over conjugation site and stoichiometry, and retention of the luciferase activity were elaborated [ 108 , 109 ]. However, the analytical application of luciferases in most cases is based on the bioluminescence of their derivatives extended with biospecific polypeptides obtained by genetic fusing.…”

“…Wouters et al reported an easily accessible, efficient, and chemoselective method for the synthesis of antibody-luciferase conjugates. [ 109 ]. The NanoLuc was genetically fused with protein G domain containing the photo-cross-linkable non-natural amino acid para-benzoylphenylalanine (developed by Hui et al [ 223 ]) that can be photo-cross-linked to the antibody using UV light illumination (365 nm).…”

“…The method does not require cloning or recombinant antibody expression and can be directly applied to almost all human and many mammalian monoclonal IgG antibodies. The obtained NanoLuc-antibody conjugates were used in cell immunostaining, solid-phase immunoassay, and Western blotting [ 109 ].…”

RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

“…The pET28a(+) vectors containing DNA encoding for Gx-SB, Gx-LB, mNG-NL and SUMO-TNFα-SB were ordered from GenScript. The sequence of mNG-NL was reported by Suzuki et al 32 , and a His-tag at the N-terminus and a Strep-tag at the C-terminus were included to facilitate purification 26 . Site-directed mutagenesis to change SB sequences was carried out using the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent technologies) and specifically designed primers.…”

“…Although methods for the generation of antibody-split luciferase fusion proteins are available, they are based on non-specific conjugation or cumbersome recombinant expression of antibody fusions 13,24 . RAPPID does not require additional protein engineering and uses protein G-mediated photoconjugation to generate well-defined antibody conjugates directly from commercially available antibodies 25,26 . Additionally, we introduce a green light-emitting calibrator luciferase as a simple method to provide a robust blue-over-green ratiometric readout and direct internal calibration.…”