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 Amersham Biosciences Application Note # 61 ❖ Page 1 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. Amersham Biosciences Application Note # 61 ❖ Page 2 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 Amersham Biosciences Application Note # 61 ❖ Page 3 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 Amersham Biosciences Application Note # 61 ❖ Page 4 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. Amersham Biosciences Application Note # 61 ❖ Page 5 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 928 East Arques Avenue Sunnyvale, CA 94086 (800) 333-5703 (408) 773-1222 Fax (408) 773-8343 ❖ Amersham Biosciences , United Kingdom (44) (1494) 793377 ❖ Amersham Biosciences , Germany (49) (2151) 83870 ❖ Amersham Biosciences , France (33) (1) 6086 6513 ❖ Amersham Biosciences , Japan All brand and product names are trademarks or registered trademarks of their respective companies. Amersham Biosciences Application Note # 61 ❖ Page 6 (81) (3) 5350-3211