A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants
- 6 February 2021
- journal article
- research article
- Published by Springer Science and Business Media LLC in Molecular Breeding
- Vol. 41 (2), 1-12
- https://doi.org/10.1007/s11032-021-01210-7
Abstract
Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics.Keywords
Funding Information
- National Transgenic Science and Technology Program (2019ZX08010003)
- National Natural Science Foundation of China (31622047)
- State Key Laboratory of Hybrid Rice
- Huazhong Agricultural University (2015RC009)
- NSF Plant Genome Research Project Grant (1740874)
- USDA National Institute of Food and Agriculture and Hatch Appropriations (1016432)
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