Abstract
Fusion tag technology is an important tool for rapid separation, purification, and characterization of proteins. Combined with monoclonal antibodies, tag epitope systems can be rapidly adapted to many assay systems. A monoclonal antibody that reacts with the matrix protein of the rabies virus CVS-11 strain was reported. The epitope (termed M) targeted by this antibody contains only six amino acids. We examine whether this specific sequence epitope can be applied as a protein tag. We show ectopic expression of M-tagged proteins has little impact on cell viability or major signaling pathways. The M tag system can be used for western blotting, immunoprecipitation, immunofluorescence staining, and flow cytometry assays. The results indicate the specificity, sensitivity, and versatility of this novel epitope tag system are comparable to the widely used FLAG tag system, providing researchers with an additional tool for molecular analysis.
Key points
• A short peptide (Pro Pro Tyr Asp Asp Asp) can be applied as a new tag.
• The new epitope-tagging fusion system has no effect on the main cellular signaling pathway.
• The epitope-tagging fusion system can be widely used for western blotting, immunoprecipitation, immunofluorescence, flow cytometry, etc.
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Data availability
The datasets generated and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Abbreviations
- A549:
-
Human non-small-cell lung cancer cell line
- CCCP:
-
Carbonyl cyanide m-chlorophenylhydrazone
- cGAS:
-
Cyclic GMP-AMP synthase
- CHX:
-
Cycloheximide
- DAPI :
-
4′,6-Diamidino-2-phenylindole
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- GFP:
-
Green fluorescent protein
- GRB2:
-
Growth factor receptor-bound protein 2
- HEK293:
-
Human embryonic kidney epithelial cell line
- HSV-1:
-
Herpes simplex virus
- IP:
-
Immunoprecipitation
- IRF3:
-
Interferon regulatory factor 3
- JC-1:
-
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimi- dazolylcarbocyanine iodide
- RIG-I:
-
Retinoic acid-inducible gene I
- SQSTM1:
-
Sequestosome 1
- STING:
-
Stimulator of interferon genes
- TBK1:
-
TANK binding kinase 1
- VACV:
-
Vaccinia virus
- VSV:
-
Vesicular stomatitis virus
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Acknowledgements
We would like to thank Dr. H.B. Shu for the generous gift of anti-STING antibody and Sean Whelan and David L. Bartlett for reporter viruses. We would like to thank the staffs from the Core Facilities of Wuhan University for their assistance with data collections.
Funding
This work was supported by the National Natural Science Foundation of China (82272336), a startup fund from Wuhan University, the Non-Profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2020-PT320-004), Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University Grant No. TFJC2018004 and Wuhan University Open Subsidy Funding Projects (LF20201347), the open funding of the Key Laboratory of Preventive Veterinary Medicine of Hubei Province (KLPVMHP-2203), and the Fundamental Research Funds for the Central Universities (2042022dx0003).
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B.F., Y.Z., Z.L., and X.C. conceived the project and designed the experiments. B.F. and X.C. supervised the project. Z.L. and B.F. wrote the manuscript. Z.L. and Y.Z. carried out and participated in the whole experiments. Q.W. participated in the whole experiment and assisted in the preparation and characterization of materials, luciferase assay, and Western blot. Z.Z., X.Y., S.Z., and L.L., supported the operation and analysis of the Western Blot and IFA. All the authors discussed the results and commentated manuscript.
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Zhang, Y., Zhao, L., Wang, Q. et al. A novel epitope tag from rabies virus has versatile in vitro applications. Appl Microbiol Biotechnol 107, 3955–3966 (2023). https://doi.org/10.1007/s00253-023-12546-9
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DOI: https://doi.org/10.1007/s00253-023-12546-9