Biomedical Engineering Reference
In-Depth Information
the luciferase gene from
G. princeps
(GLuc) fused to the
C. albicans
PGA59
(GPI-linked protein required for cell wall integrity) (Moreno-Ruiz et al.
2009, Tannous et al. 2005). In this way, the luciferase is exposed at the fungal
surface and it is more accessible to its substrate (Enjalbert et al. 2009).
New Promising Applications of Bioluminescent Proteins—
Protein Interactions
In vivo
analysis and imaging of interacting proteins constitutes another
application of bioluminescent proteins. In 2005, Kadurugamuwa and
colleagues used multicolour bioluminescence to monitor both, disease
process (bacterial spread) and host response (neural injury), at the same
time in the context of a living organism. For this purpose, this author
combined a mouse transgenic model expressing luciferase from fi refl y
fused to the GFAP (a host glial protein) promoter and an engineered
bacterium (
Streptococcus pneumoniae
) for bioluminescence (
lux
) for
simultaneous
in vivo
analysis of disease progression and the GFAP
response (Kadurugamuwa et al. 2005). It is important to remark new
studies focused in protein interaction by using multicolour luminescent
proteins. Recently, Hida and colleagues showed an improved imaging
method for spatiotemporal analysis of different protein interactions. For
this purpose, the authors used multicolour luciferases with different
spectral characteristics and developed protein fragments complementation
assays, a technique based in bimolecular fl uorescence complementation
(BiFC) (Hida et al. 2009). They developed a novel luciferase fragment
by mutagenesis in order to cross complement intra- and inter-luciferase
frangments (from fi refl y and click beetle) with high effi ciency. Finally, the
authors validated the system by assaying different protein interactions
either
in vitro
or
in vivo
.
CONCLUSION
Bioluminescence gene reporters have found general use in the analysis
of fungal genes. Although bioluminescence has the dissadvantage over
fl uorescence that fl ow cytometry can not be used to monitor individual
cells, its superior signal-to-noise values allow the precise quantifi cation
of transcriptional activity inside the cells. A common problem with these
types of studies derive also from the permeability of the substrates inside
the fungal cell; however, the development of new substrates and the genetic
engineering to express luciferase in cell surface may easily circumvent
this problem. It is envisioned that the development by molecular genetics
of new luciferases will allow to monitor time-dependent events within
