Biomedical Engineering Reference
In-Depth Information
time. Expression of gene fusion products usually takes place over a period of
several hours after transfection and continues for 72-96 h after introduction
of plasmid DNA into mammalian cells. In many cases, the plasmid DNA can
be incorporated into the genome in a permanent state to form stably trans-
formed cell lines. The choice of transient or stable transfection depends upon
the target objectives of the investigation.
Structure of GFP
There are several reasons why GFP is a powerful biomarker. GFP is easy to
detect by its fluorescence in cells; therefore, it is more versatile than most
other bioluminescent molecules that require the addition of other substances
before they glow. For example, firefly luciferase requires ATP, magnesium,
and luciferin before it luminesces. It is a fairly small and compact protein
(molecular weight, 27 kDa). The small size does not hinder the proper func-
tion of the protein to which it is attached, and particularly its intracellular
tra
cking and translocation. Among the most important aspects of the GFP
to appreciate is that the entire 27 kDa native peptide structure is essential to
the development and maintenance of its fluorescence. It is remarkable that the
fluorophore derives from a triplet of adjacent amino acids: the serine, tyrosine,
and glycine residues at locations 65, 66, and 67 (referred to as Ser65, Tyr66,
and Gly67; in Fig. 10.3).
Gly 67
Tyr 66
+ 2H
+
−
H
2
O
Ser 65
395 nm
Imidazolinone
Ring System
H
+
−
Tyr 66
Prematuration
Chromophore
Cyclization
Reaction Sites
Quinone
475 nm
Fig. 10.3.
GFP-chromophore and the
β
-barrel structure. Within the hydrophobic
environment in the center of the GFP, a reaction occurs between the carboxyl car-
bon of Ser65 and the amino nitrogen of Gly67 that results in the formation of an
imidazolin-5-one heterocyclic nitrogen ring system. Further oxidation results in con-
jugation of the imidazoline ring with Tyr66 and maturation of a fluorescent species.
It is important to note that the native GFP fluorophore exists in two states. A pro-
tonated form that has an excitation maximum at 395 nm, and a unprotonated one
that absorbs at approximately 475 nm. However, regardless of the excitation wave-
length, fluorescence emission has a maximum peak wavelength at 507 nm, although
the peak is broad and not well defined
