APPLICATION NOTE
#61
Green Fluorescent Protein Applications
Introduction
Green fluorescent protein (GFP) is widely used as a reporter
molecule for the study of protein localization, protein binding
events, and gene expression. Using recombinant DNA
technology, the coding sequence for GFP can be spliced with
that of other proteins to create fluorescent fusion proteins. GFP
fusion proteins can be used in vivo to localize proteins of interest
to specific cell types and subcellular locations. They can also be
used in vitro to study protein-protein interactions. In geneexpression studies, when GFP expression is placed under the
control of a specific promoter or DNA regulatory sequence,
GFPs serve as reporters of transcriptional activity. GFP provides
unique advantages as a reporter molecule in these applications
including the capacity for expression in many different cell
types and organisms without the need for additional substrates
or cofactors. Fluorescence from GFP is direct, stable, and readily
detectable using common modes of fluorescence detection.
Structural and Spectral Properties
GFP is isolated from the jellyfish Aequorea victoria, found off the
coast of the northwest United States1. In A. victoria, light is
produced as a result of energy transfer from the Ca2+-activated
photoprotein aequorin to GFP2. GFP is comprised of 238 amino
acids with a molecular weight of 27 kDa. The chromophore
responsible for the light emission is a hexapeptide within the
protein. The cDNA for GFP has been cloned and sequenced,
permitting the creation of engineered GFP variants3.
Wild-type GFP has a major excitation peak at 395 nm (minor
peak at 475 nm) and emits bright green light with an emission
peak at 508 nm. Traditionally, fluorescence from GFP-expressing
cells is produced using ultraviolet-light (UV) excitation and
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detected by fluorescence microscopy or by fluorescenceactivated cell sorting (FACS). This application note describes
the use of the Amersham Biosciences FluorImager® and Storm®
systems for the visualization and quantitation of GFP
fluorescence in expression analysis and in the study of proteinprotein interactions.
GFP Variants
Wild-type GFPs are not optimal for some reporter-gene
applications. When excited by blue light common to
fluorescence microscopy and FACS, such as the 488-nm
argon-ion laser, the fluorescence intensity from wild-type GFPs
is relatively low. In addition, a significant lag in the development
of fluorescence after protein synthesis can occur and complex
photoisomerization of the GFP chromophore may result in the
loss of fluorescence. Furthermore, wild-type GFPs are expressed
at low levels in many higher eukaryotes 4. Numerous GFP
variants 3 have been engineered to improve upon these
limitations.
Several GFP variants are available with a significantly larger
extinction coefficient for excitation at 488 nm and a modified
gene sequence with codon usage preferentially found in highly
expressed eukaryotic proteins. The combination of improved
fluorescence intensity and higher expression levels yields an
enhanced GFP, which provides greater sensitivity in most
systems. The detection limits for purified wild-type GFP, EGFP,
GFP-S65T, and GFPuv proteins (Table 1) were determined in gel
electrophoresis using both the FluorImager and Storm systems.
GFP-S65T, EGFP, and GFPuv were best suited for analysis
using the FluorImager (488-nm excitation) and could be detected
at about 0.3ng per band in polyacrylamide gels. The GFP-S65T
and EGFP variants were also the most sensitive GFPs when
imaged using the Storm blue-fluorescence mode, although the
Storm limit of detection for these two was about 8ng. Storm
detection of the wild-type protein was limited to approximately
15ng. With the FluorImager system, wild-type GFP was also
found to be less compatible than the other variants tested and
was detected at levels of about 2ng per band. The linear range
of detection for each GFP was between 1.5 and 3 orders of
magnitude.
Peak Excitation (nm) Peak Emission (nm) Codon Optimization
Wild-Type GFP
395/470
509/540
None
Red-Shifted:
EGFP
GFP-S65T
488
488
507
511
Human
Table 1.
GFPuv
395
509
E.coli
GFP and GFP variants (CLONTECH)
with characteristics spectral properties
and codon optimization.
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Detection of GFP Using FluorImager and Storm Systems
Monitoring Gene Expression in Yeast and Bacteria
The FluorImager SI was used to analyze transient gene
expression in transformed yeast cells that used GFP as the
reporter. The GFP-transformed colonies were spotted and
grown on agar plates. Expression of GFP was observed by
scanning the agar plate using 488-nm excitation (Figure 1).
The blue-fluorescence mode of the Storm 860 was used to
analyze E.coli cells transformed with a GFP expression vector.
Following overexpression of the fusion protein, cell lysates
were analyzed by SDS-PAGE (Figure 2). Heat treatment of the
lysate prior to SDS-PAGE was done at a reduced temperature
to allow direct detection of GFP fluoroscence. Denaturing
conditions, such as boiling, result in loss of fluoroscence. The
GFP fusion protein is detected in as little as 2 µl of the bacterial
cell lysate.
Figure 1.
Varying expression levels from
GFP-reporter constructs in yeast
colonies. Colonies were spotted and
grown on agar plates with incubation
at 37°C. The agar plate was placed in
a microplate tray and scanned with a
FluorImager SI at a PMT setting of
575 volts and a resolution of 100 µm.
The image was kindly provided by
Drs. John Phillips and Matt Ashby,
Acacia Biosciences, Richmond, CA.
Figure 2.
The vector for PBAD-GFP, a GFPuv
variant (Maxygen, Santa Clara, CA),
was cloned and overexpressed in E.coli.
The cells were lysed with 5 mg/ml
lysozyme and 25 mM EDTA at room
temperature for 10 minutes, followed
by treatment with 100 mg/ml DNase
in 35 mM MgCl2 for 15 minutes at
room temperature. Lysate samples from
2 to 15 µ l were heated at 45°C for
3 minutes and analyzed in a 10% SDS
polyacrylamide gel. The gel was
scanned using the Storm 860 bluefluorescence mode (450+30nm) with
a PMT setting of 1000 volts and a
resolution of 200 µm. The image was
kindly provided by Dr. Wai-Mun
Huang, University of Utah, Salt Lake
City, UT.
15 µl
8
4
2
2
4
6
8
Cell Lysate
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Study of Protein-Protein Interactions
When used as a probe in a fusion protein, GFP functions as an
independent domain without altering the properties of the
protein of interest. As such, GFP and its variants are effective
tools for in vivo and in vitro functional analyses of proteinprotein interactions. GFP has been used to demonstrate
interaction between the S-peptide and S-protein fragments of
ribonuclease A5. S15 DNA and codons of six histidine residues
were added, respectively, to the 5’- and 3’-ends of the cDNA
that encode the S65T~GFP mutant (peak excitation at 488-nm)
to produce a S15~GFP(S65T)~His6 fusion protein. A fluorescent
gel retardation assay was run by incubating varying amounts
of S-protein with purified S15 peptide~GFP(S65T)~His6 and
resolving the complexes from the free components in a native
polyacrylamide gel (Figure 3). The 488-nm excitation source of
a FluorImager SI system was used to acquire the image.
In another study, fusion proteins created between calmodulin
(CaM) or calmodulin-like protein (CLM) and the GFP~S65T
variant were used in a “gel overlay” assay to rapidly screen for
interacting proteins6. Figure 4 shows rat brain protein fractions
resolved in SDS-polyacrylamide gels that were transferred to
membranes and incubated with the GFP fusion proteins.
Imaging of the membranes using the blue-fluorescence mode
of a Storm 840 reveals GFP fluorescence at sites where CaM or
CLM have bound to specific target proteins.
0.95
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0
nM S-protein
Bound
Free
2
1
µg
µg
KDa
Mw 86 23 12 2.4
Mw 86 23 12 2.4
KDa
250
250
148
148
60
60
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Figure 3.
GFP gel retardation assay used to
quantify the interaction between
S-protein and S15~GFP(S65T)~His6
using the FluorImager SI. A constant
amount of S15~GFP(S65T)~His6
(1 mM) was incubated for 20 minutes
with varying amounts (0-0.95 µM) of
S-protein at 20°C in 10 mM Tris-HCl
(pH 7.5) that contains 5% glycerol.
Electrophoresis was performed on a
native 6% polyacrylamide gel. After
electrophoresis, the gel was scanned
with a FluorImager SI. The image was
kindly provided by Sang-Hyun Park
and Ronald Raines, University of
Wisconsin, Madison, Wisconsin.
Figure 4.
GFP gel overlay assay used to study
protein-protein interactions using
GFP(S65T)-CaM (panel 1) and
GFP(S65T)-CLP (panel 2) as probes.
Rat brain synaptosomal protein
fractions (2.4 to 86 µg) were prepared
and varying amounts were resolved
on a 6.5% polyacrylamide gel. After
protein transfer to PVDF, membranes
were probed with one of the GFP fusion
proteins. The blue-fluorescence mode
of the Storm 840 was used with a
PMT setting of 800 volts. The image
was kindly provided by Dr. Nandor
Garamzsegi, Mayo Clinic, Rochester,
MN.
Conclusion
GFP is an extremely valuable tool for monitoring the dynamic
processes of living cells and organisms. Direct detection and
quantitation of GFP is feasible using both the Storm 840/860
and FluorImager systems. The 488-nm excitation source in the
FluorImager and the blue-fluorescence mode of the Storm
provide efficient excitation of GFP and GFP variants. Note that
as an alternative to direct visualization of GFP, antibodies
against GFP are available. In applications where direct detection
of GFP does not provide sufficient signal (such as Western
blotting and immunoprecipitation), indirect detection using an
enzyme-conjugated anti-GFP provides a means for signal
amplification.
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References
1. Chalfie, M. et al., Green fluorescent
protein as a marker for gene expression. Science (1994) 263: 802-805.
2. Inouye, S. and F.I. Tsuji, Aequorea
Green fluorescent protein: Expression of the gene and fluorescence
characteristics of the recombinant
protein. FEBS letter (1994) 341:
277-280.
3. Heim, R. et al., Wavelength
mutations and posttranslational
autoxidation of green fluorescent
protein. Proceedings of National
Academy of Sciences, USA (1994)
91:12501-12504.
4. Kain, S.R. et al., Green fluorescent
protein as a reporter of gene expression and protein localization.
BioTechniques (1995) 19, No. 4:
650-655.
5. Park, S-H and R.T. Raines, Green
fluorescent protein as a signal for
protein-protein interactions. Protein
Science. (1997) 6: 2344-2349.
6. Garamszegi, N. et al., Application of
a chimeric Green fluorescent protein
to study Protein-Protein interactions.
BioTechniques. (1997) 23: 864-872.
Author
Anu Kondepudi,
Amersham Biosciences , Inc.
Amersham Biosciences
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