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Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein–Protein Interactions and Cellular Protein Complex Localizations

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Abstract

Investigations of protein–protein interactions (PPIs) are of paramount importance for comprehending cellular processes within biological systems. The bimolecular fluorescence complementation (BiFC) assay presents a convenient methodology for visualizing PPIs within live cells. While a range of fluorescent proteins have been introduced into the BiFC system, there is a growing demand for new fluorescent proteins to accommodate the expanding requirements of researchers. This study describes the introduction of Tagged blue fluorescent protein 2 (TagBFP2) into the BiFC assay to verify the interaction between two proteins, with Enhanced yellow fluorescent protein (EYFP) employed as a positive control. Both fluorescent proteins demonstrated optimal performance in this study. Compared to EYFP, the BiFC system utilizing TagBFP2 yielded a higher signal-to-noise ratio, which facilitated differentiation of the signal of PPIs from noise and enabled employment of other fluorescent proteins within the BiFC assay. Notably, the utilization of a fluorescent secondary antibody in immunofluorescence applications or the tagging of an interest protein with a fluorescent protein occupied the green or yellow channel. Overall, the present article introduces a BiFC assay that is highly straightforward, reliable, and replicable, with the ability to be completed within 1 week. This method requires neither expensive instrumentation nor technical skills of a high order.

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Data Availability

All data generated or analyzed during this study are included in the manuscript.

Abbreviations

BiFC:

Bimolecular fluorescence complementation

PPIs:

Protein–protein interactions

Co-IP:

Co-immunoprecipitation

FRET:

Fluorescence resonance energy transfer

GFP:

Green-fluorescent proteins

bZIP:

Basic region-leucine zipper

AP-1:

Activator protein-1

cDNA:

Complementary DNA

DMEM:

Dulbecco’s modified Eagle’s medium

EYFP:

Enhanced yellow fluorescent protein

TagBFP2:

Tag blue fluorescent protein 2

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Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (82273278, 82002630, 81874215), Shanghai Association for Science and Technology (201409003000, 201409002400 and 20YF1426200), Outstanding Leaders Training Program of Pudong Health Committee of Shanghai (PWRl2017-03), Pudong Science and Technology Development Fund (pkj2019-y35).

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Author notes

  1. Zhonggang Shi and Xing Gao have contributed equally to this work.

Authors and Affiliations

  1. Brain Injury Center, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, No. 160 Pujian Road, Pudong District, Shanghai, 200127, People’s Republic of China

    Zhonggang Shi, Wenrui Zhang, Binghong Chen, Mengying Wang, Keman Liao, Yongming Qiu & Yingying Lin

  2. Shanghai Cancer Institute, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, No. 160 Pujian Road, Pudong District, Shanghai, 200127, People’s Republic of China

    Zhonggang Shi, Wenrui Zhang, Mengying Wang & Yingying Lin

  3. Department of Medical Oncology, Guangxi Medical University Cancer Hospital, Nanning, 530021, Guangxi, People’s Republic of China

    Xing Gao

  4. Department of Neurosurgery, Shanghai Pudong Hospital, Fudan University, Shanghai, 201399, People’s Republic of China

    Zhihan Wang & Li Ren

  5. Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Yueyang Road 320, Shanghai, 200031, China

    Yujia Zhai

  6. Department of Neurosurgery, Xinhua Hospital Affiliated to Shanghai JiaoTong University School of Medicine, No. 1665 Kongjiang Road, Shanghai, 200092, People’s Republic of China

    Xuhui Wang

  7. Department of Neurosurgery, Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai, 202150, People’s Republic of China

    Xuhui Wang

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  1. Zhonggang Shi
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Contributions

YL designed the structure of the manuscript. ZS, XG, BC, MW, KL, ZW, and YZ contributed to the performance of experiments, ZS, XG, WZ, LR, YQ and XW contributed to the written and revision sections to the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xuhui Wang or Yingying Lin.

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Shi, Z., Gao, X., Zhang, W. et al. Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein–Protein Interactions and Cellular Protein Complex Localizations. Mol Biotechnol (2023). https://doi.org/10.1007/s12033-023-00860-6

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