Biomedical Engineering Reference
In-Depth Information
variants of the yeast two-hybrid assay, are extremely useful
techniques. However, typically none of these techniques reveal
the cellular localization where the protein interaction normally
takes place. In the case of immunoprecipitation and pull-down
experiments, cells need to be broken, and the assays are performed
under in vitro conditions. Such conditions do not favor preserva-
tion of weak protein-protein interactions that nevertheless are
typical for regulatory molecules. Different two-hybrid interaction
assays usually require that the interaction occurs at a predeter-
mined site within the cell such as the nucleus. A complicating
feature in these assays is that all cofactors needed for an effi cient
protein interaction to take place may not be present in this unnat-
ural site of interaction.
Development of green fl uorescent protein (GFP) technology
has enabled visualization of protein localization and movement
dynamics under in vivo conditions [
1
]. However, the visualiza-
tion of the total localization pattern of GFP-tagged target pro-
tein does not reveal where within its overall distribution the
proteins interact with different binding partners. GFP technol-
ogy has recently been utilized in two in vivo protein interaction
methods that can be used to reveal the cellular site of interac-
tion: the fl uorescence resonance energy transfer (FRET) and
bimolecular fl uorescence complementation (BiFC) [
2
,
3
]). In
FRET, the difference in fl uorescence signal or its lifetime is
detected when the fl uorophores are close enough in the same
complex [
4
]. In contrast, in BiFC, a fl uorescence signal is gener-
ated only when nonfl uorescent fragments of GFP are brought
together by interaction of the target molecules fused to GFP
fragments. Thus, BiFC is potentially very sensitive with a low
background. The drawback of BiFC is that it does not allow
interaction dynamics studies as an assembly of the GFP results in
signifi cant stabilization of the target protein-GFP complexes [
2
,
5
].
Recently, BiFC has been shown to function in various cells from
bacteria to mammals [
5
,
6
]. At the moment, there exist several
versions of BiFC that make use of the different spectral proper-
ties of GFP variants. Protein interactions have been visualized
with the help of fragments of enhanced yellow fl uorescent pro-
tein (EYFP) or its variants Venus and Citrine, cyan fl uorescent
protein (CYFP) or its variant Cerulean, and blue fl uorescent
protein (BFP) and monomeric red fl uorescent protein (mRFP)
fragments [
7
-
9
]. Combinations of these variants enable simulta-
neous visualization of several protein interactions [
10
].
In this chapter, we report the method and tools for the use of
BiFC system with EYFP fragments in the yeast
S. cerevisiae
and
Venus fragments in mammalian cells.
