Biology Reference
In-Depth Information
Table 6.1 Pros and cons of genetically encoded versus fluorescent peptide biosensors
Genetically encoded fluorescent
biosensors
Fluorescent peptide/protein biosensors
Features
Features
Genetically
encoded
constructs
Peptide or protein backbone onto
which a synthetic fluorescent probe
is introduced during or postsynthesis
through
based on two AFPs
Essentially FRET/FLIM
chemical
or
enzymatic
conjugation
Environmentally sensitive, chelation-
enhanced, self-reporting
Pros
Pros
Easy to engineer through
molecular biology approaches
Easy to transfect or microinject
into cultured cells
Small size
Versatility in design and engineering
Easy and cheap to synthesize or
express through recombinant protein
engineering
Allow control over concentration and
time of addition to target or cells
Allow technological improvements:
introduction of quenchers, amino acid
caging
Cons
Cons
Large size—essentially due to the
size of the autofluorescent proteins
Require ectopic expression
Lack of control over expression
levels and timing
Heterogeneous expression in a
population of cells
Difficult to obtain optimal
expression for
in vivo
imaging
Require means for efficient introduc-
tion into living cells and
in vivo
Require choice of NIRF probes for
in vivo
applications
when the two chains are brought together for a protein/protein interaction;
(iii) biosensors based on bimolecular fluorescence complementation (BiFC),
in which two fragments of a split AFP are brought together as a consequence
of the recognition event, thereby reconstituting the intact and fully
fluorescent protein; (iv) single-chain biosensors which bear a single AFP
whose spectral properties change in response to the recognition of a
target by an encoded element other than the AFP; and (v) biosensors
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