TY - JOUR
T1 - Versatile protein tagging in cells with split fluorescent protein
AU - Kamiyama, Daichi
AU - Sekine, Sayaka
AU - Barsi-Rhyne, Benjamin
AU - Hu, Jeffrey
AU - Chen, Baohui
AU - Gilbert, Luke A.
AU - Ishikawa, Hiroaki
AU - Leonetti, Manuel D.
AU - Marshall, Wallace F.
AU - Weissman, Jonathan S.
AU - Huang, Bo
N1 - Funding Information:
We thank Yuichiro Miyaoka, Amanda Chan and Bruce Conklin for helping with TaqMan/ddPCR assay, and Wei Qiang Ong for the CD86-SNAP-tag vector and the b2AR vector. This work is supported by NIH DP2OD008479 (to B.H.), R21MH101688 (to B.H. and D.K.), R01DA036858 (to L.A.G., M.D.L. and J.S.W.), R01GM097017 (to H.I. and W.F.M.), the W.M. Keck Foundation (to J.H., B.C. and B.H.). S.S. receives support from the Japan Society for the Promotion of Science postdoctoral fellowship for research abroad.
PY - 2016/3/18
Y1 - 2016/3/18
N2 - In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
AB - In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
UR - http://www.scopus.com/inward/record.url?scp=84961654781&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84961654781&partnerID=8YFLogxK
U2 - 10.1038/ncomms11046
DO - 10.1038/ncomms11046
M3 - Article
C2 - 26988139
AN - SCOPUS:84961654781
VL - 7
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
M1 - 11046
ER -