RRC ID 49532
Author Shimozono S, Miyawaki A.
Title Engineering FRET constructs using CFP and YFP.
Journal Methods Cell Biol.
Abstract Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Here we discuss how to engineer constructs for FRET between the cyan- and yellow-emitting variants of green fluorescent protein (GFP) from Aequorea victoria (CFP and YFP, respectively). Throughout this chapter, we stress the fact that FRET is highly sensitive to the relative orientation and distance between the donor and the acceptor. The chapter consists of two parts. First, we discuss FRET-based indicators encoded by single genes, which were developed in our laboratory. In this approach, a number of different constructs can be made for a comparative assessment of their FRET efficiencies. For example, the length and sequence of the linker between the fluorescent protein and the host protein should be optimized for each specific application. In the second part, we describe the use of long and flexible linkers for engineering FRET constructs, including an introduction to a general and efficient tool for making successful fusion proteins with long and flexible linkers. When CFP and YFP are fused through floppy linkers to two protein domains that interact with each other, the two fluorescent proteins will associate due to the weak dimerization propensity of Aequorea GFP, which results in moderate FRET. This approach has become even more powerful due to the construction of a new pair of fluorescent proteins for FRET: CyPet and YPet.
Volume 85
Pages 381-93
Published 2008
DOI 10.1016/S0091-679X(08)85016-9
PII S0091-679X(08)85016-9
PMID 18155471
MeSH Base Sequence Fluorescence Resonance Energy Transfer / methods* Green Fluorescent Proteins* HeLa Cells Humans Luminescent Proteins* Microscopy, Confocal / methods* Microscopy, Fluorescence / methods* Molecular Sequence Data Recombinant Fusion Proteins* Transfection
IF 1.588
Resource
DNA material pBS coupler 1 (RDB15126) pBS coupler 2 (RDB15127) pBS coupler 3 (RDB15128) pBS coupler 4 (RDB15129) pBS coupler 5 (RDB15130) pBS coupler 6 (RDB15131).