Keith V. Wood, Ph.D.,Head of Research, Promega Corporation

NanoBiT Complementation Reporter Provides Accurate Assessment of Intracellular Protein Interactions under Physiological Conditions

Keith V. Wood, Ph.D.,Head of Research, Promega Corporation

Aula Cappa, Monday November 9th 2:30 p.m.

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. Using a combination of biochemical and cellular models, we have demonstrated that NanoBiT accurately reflects the binding affinity and association kinetics of intracellular protein interactions. Moreover, the response dynamics are robust to experimental conditions, providing reliable data over a broad range of temperatures and gene expression levels. Our characterization of the intrinsic properties of NanoBiT establish its general suitability for analysis of intracellular protein interactions under physiological conditions.

Dr. Keith Wood is Head of Research, Advanced Technologies and Senior Research Fellow at Promega Corporation. Widely regarded for his work in bioluminescence, he currently leads a cross-disciplinary team focused on long-range innovation in biochemical and cellular research.

His current research centers on the development of novel bioanalytical capabilities, including novel bioluminescent chemistries, intracellular detection technologies, and efficient isolation methods for protein and drug complexes.

He has authored over 55 journal articles and book chapters and is an inventor for 83 issued and 99 pending U.S. and foreign patents.

Dr. Wood received his Ph.D. in Biochemistry at University of California-San Diego, where he also performed post-doctoral research before joining Promega in 1990.

Select Publications:

1. A luminescent assay for real-time measurements of receptor endocytosis in living cells. Robers MB, Binkowski BF, Cong M, Zimprich C, Corona C, McDougall M, Eggers CT, Hartnett J, Machleidt T, Fan F. Wood KV. Anal Biochem. 2015 Nov 15;489:1-8. doi: 10.1016/j.ab.2015.08.005. Epub 2015 Aug 13.
2. Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag. Friedman Ohana R, Kirkland TA, Woodroofe CC, Levin S, Uyeda HT, Otto P, Hurst R, Robers MB, Zimmerman K, Encell LP, Wood KV. ACS Chem Biol. 2015 Aug 6.
3. Application of BRET to monitor ligand binding to GPCRs.Stoddart LA, Johnstone EK, Wheal AJ, Goulding J, Robers MB, Machleidt T, Wood KV, Hill SJ, Pfleger KD. Nat Methods. 2015 Jul;12(7):661-3. doi: 10.1038/nmeth.3398. Epub 2015 Jun 1.
4. NanoBRET—A Novel BRET Platform for the Analysis of Protein-Protein Interactions. Machleidt T, Woodroofe CC, Schwinn MK, Méndez J, Robers MB, Zimmerman K, Otto P, Daniels DL, Kirkland TA, Wood KV. ACS Chem Biol. 2015 Aug 21;10(8):1797-804. doi: 10.1021/acschembio.5b00143. Epub 2015 Jun 9.
5. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV. ACS Chem Biol., 2012 Nov 16; 7(11):1848-57.