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Fluorescence complementation via EF-hand interactions

Paper ID Volume ID Publish Year Pages File Format Full-Text
24541 43525 2009 9 PDF Available
Title
Fluorescence complementation via EF-hand interactions
Abstract

Fluorescence complementation technology with fluorescent proteins is a powerful approach to investigate molecular recognition by monitoring fluorescence enhancement when non-fluorescent fragments of fluorescent proteins are fused with target proteins, resulting in a new fluorescent complex. Extension of the technology to calcium-dependent protein–protein interactions has, however, rarely been reported. Here, a linker containing trypsin cleavage sites was grafted onto enhanced green fluorescent protein (EGFP). Under physiological conditions, a modified fluorescent protein, EGFP-T1, was cleaved into two major fragments which continue to interact with each other, exhibiting strong optical and fluorescence signals. The larger fragment, comprised of amino acids 1–172, including the chromophore, retains only weak fluorescence. Strong green fluorescence was observed when plasmid DNA encoding complementary EGFP fragments fused to the EF-hand motifs of calbindin D9k (EF1 and EF2) were co-transfected into HeLa cells, suggesting that chromophore maturation and fluorescence complementation from EGFP fragments can be accomplished intracellularly by reassembly of EF-hand motifs, which have a strong tendency for dimerization. Moreover, an intracellular calcium increase upon addition of a calcium ionophore, ionomycin in living cells, results in an increase of fluorescence signal. This novel application of calcium-dependent fluorescence complementation has the potential to monitor protein–protein interactions triggered by calcium signalling pathways in living cells.

Keywords
EGFP; Fluorescence complementation; EF-hand; Calcium-dependent; Protein–protein interactions; EGFP fluorescent fragment
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Fluorescence complementation via EF-hand interactions
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 142, Issues 3–4, 15 July 2009, Pages 205–213
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us