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Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

Published on: May 17, 2010

Versatile protein tagging in cells with split fluorescent protein.

Daichi Kamiyama¹, Sayaka Sekine¹, Benjamin Barsi-Rhyne²

¹Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.

Nature Communications

|March 19, 2016

Summary

Researchers developed novel split fluorescent protein tags (FP11-tags) for live cell protein imaging. These small epitope tags minimize functional impact and offer versatile applications in imaging and protein studies.

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Area of Science:

Cell Biology
Molecular Imaging
Biotechnology

Background:

Epitope tags are increasingly used for live cell protein imaging due to their small size, which can reduce functional perturbation and allow signal amplification.
However, epitope tags can suffer from background noise, limiting their effectiveness.

Purpose of the Study:

To adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labeling, addressing background issues and enhancing imaging capabilities.
To demonstrate the utility of these FP11-tags in various biological applications.

Main Methods:

Developed GFP11 and sfCherry11 tags derived from split super-folder fluorescent proteins.
Inserted FP11-tags into endogenous genomic loci using CRISPR-mediated homology-directed repair.
Utilized tandem arrangements of FP11-tags for enhanced fluorescence and reduced photobleaching.

Main Results:

FP11-tags enabled cost-effective and scalable live cell protein labeling with reduced background.
Tandem FP11-tags improved fluorescence signal for tracking intraflagellar transport particles.
Demonstrated reduced photobleaching for live microtubule imaging using tandem tags.
Showcased the utility of tandem GFP11-tag in scaffolding protein oligomerization.

Conclusions:

FP11-tags offer a versatile labeling tool for live cell imaging, overcoming background limitations of traditional epitope tags.
These tags are valuable for both imaging and non-imaging applications, including protein multimerization control.