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Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility 1,2,3,4 or may not work in living cells 5,6,7 . Green fluorescent protein (GFP) fusions can misfold 8 or exhibit altered processing 9 . Fluorogenic biarsenical FLaSH or ReASH 10 substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization 10 . An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP 11 , but the GFP fragments reported thus far are large and fold poorly 11,12 , require chemical ligation 13 or fused interacting partners to force their association 11,12,13,14 , or require coexpression or co-refolding to produce detectable folded and fluorescent GFP 11,12 . We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Biotechnology Springer Journals

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

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References (42)

Publisher
Springer Journals
Copyright
Copyright © 2004 by Nature Publishing Group
Subject
Life Sciences; Life Sciences, general; Biotechnology; Biomedicine, general; Agriculture; Biomedical Engineering/Biotechnology; Bioinformatics
ISSN
1087-0156
eISSN
1546-1696
DOI
10.1038/nbt1044
Publisher site
See Article on Publisher Site

Abstract

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility 1,2,3,4 or may not work in living cells 5,6,7 . Green fluorescent protein (GFP) fusions can misfold 8 or exhibit altered processing 9 . Fluorogenic biarsenical FLaSH or ReASH 10 substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization 10 . An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP 11 , but the GFP fragments reported thus far are large and fold poorly 11,12 , require chemical ligation 13 or fused interacting partners to force their association 11,12,13,14 , or require coexpression or co-refolding to produce detectable folded and fluorescent GFP 11,12 . We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.

Journal

Nature BiotechnologySpringer Journals

Published: Dec 5, 2004

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