Protocol for Quantitation of GFP and GFP Variants

1. Introduction

The green-fluorescent protein (GFP) from the jellyfish Aequorea victoria is used as a fluorescent marker for gene expression in a variety of organisms ranging from bacteria to higher plants and animals.(1) The cloning of the gene for GFP (2, 3) and its subsequent expression in heterologous systems(1, 3, 4) has established GFP as a unique genetic reporter system. It has become useful for monitoring gene expression in vivo, in situ, and in real time. When expressed in either eukaryotic or prokaryotic cells, GFP gives forth a bright green fluorescence.

Figure 1. GFP emits green light (absorbance maximum at 509 nm) when excited with blue or UV light (absorbance maximum at 395nm). (1)

Unlike other bioluminescent reporters, GFP fluoresces in the absence of any other intrinsic or extrinsic proteins, substrates, or cofactors. Fluorescence is stable, species-independent, and can be monitored noninvasively in living cells and whole animals, such as in the case of transparent organisms.(1)

Purified GFP (from either native or recombinant sources) is a 27-kDa monomer consisting of 238 amino acids(2) . In the bioluminescent jellyfish Aequorea victoria, light is produced when energy is transferred from the Ca2+ activated photoprotein aequorin to GFP.(5, 6, 7). After being formed, the protein appears to undergo an autocatalytic reaction creating a fluorophore(8) from a backbone sequence of amino acids, near residue number 66, which is a tyrosine.

A putative GFP chromophore structure was described in 1979, and has since been modified(8, 5) (see Figure 2). This structure is unique amongst natural chromophores, comprising a cyclic tripeptide sequence, serine-dehydrotyrosine-glycine, which is covalently linked through the protein's peptide backbone. The mechanism of chromophore formation is unknown, but oxidation of tyrosine to dehydrotyrosine is probably an essential step that occurs gradually after the formation of the protein.

Figure 2. The Green-Fluorescent protein chromophore.(8)

2. Materials Required

• TD-700 Fluorometer with standard PMT (P/N 7000-009)
• 13 mm round test tube adaptor (P/N 7000-981)
• 13 mm x 100 mm borosilicate glass test tubes (P/N 10-031)
• Tris-EDTA buffer pH 8.00 (10 mM tris/10 mM EDTA/0.02% Sodium Azide (w/v), adjusted to pH 8.00)
• 20µg solution of recombinant Aequorea Green-Fluorescent Protein from CLONTECH Laboratories, Inc. Telephone (415)424-8222 (catalogue # 8360-1)
• GFP (395 nm ex, 509 nm em) and GFPuv (360 - 400 nm ex, 509 nm em): Near UV Mercury Vapor lamp (P/N 10-049), 390 nm excitation filter (P/N 034-0390), 510 nm - 700 nm emission filter (P/N 10-109R-C)
• EGFP (490 nm ex, 505 nm em) and EYFP (488 nm ex, 527 nm em): Blue Mercury Vapor lamp (P/N 10-089), 486 nm excitation filter (P/N 034-0486), 510 nm - 700 nm emission filter (P/N 10-109R-C)
• EBFP (380 nm ex, 440 nm em): Near UV Mercury Vapor lamp (P/N 10-049), 390 nm excitation filter (P/N 034-0390), 410 nm - 600 nm emission filter (P/N 10-110R-C)

3. Standard preparation

3.1 The first step in quantitating GFP in solution is to prepare a standard solution of highly purified recombinant GFP.

3.2 A 20µg aliquot of highly purified recombinant GFP is diluted into a final volume of 4 ml with 10 mM Tris-EDTA buffer (TE) containing azide at pH 8.00 to produce a 0.005 mg/ml standard solution (5µg/ml).

3.3 This is placed into a labeled 13 x 100 mm borosilicate test tube and sealed with parafilm. Solutions of this concentration are routinely used in the GFP field and are quite suitable to calibrate fluorometers such as the TD-700.

4. Fluorometer Calibration

4.1 Considerations

4.1.1 Upon receiving and unpacking your TD-700 you must prepare it for GFP quantitation. Identify the operating manual and keep it within reach throughout this operation.

4.1.2 Follow the procedure outlined in your operation manual to install the near UV mercury vapor lamp which is shipped outside the unit.

4.1.3 Install your GFP filters into the filter cylinder and place it into the fluorometer (remember to wipe any fingerprints off of any filter or cuvette after you handle it). It will help to use the diagrams in your TD-700 manual as a guide. The ports for each set of filters are labeled EX for excitation and EM for emission and each pair of ports is labeled A through D. Choosing one pair of ports, carefully insert your filters. The 390 nm filter has a reflective face which should be installed so that it will face out towards the lamp. Each filter is held in place by a circular rubber grommet, or o-ring.

4.1.4 At the ends of the filter cylinder are labeled marks corresponding to the pair of filter ports you have chosen. Insert the filter cylinder into the fluorometer while aligning this mark with the silver alignment mark found on the inside rim of the fluorometer's sample chamber.

4.2 Calibration

4.2.1 Close the TD-700 lid and turn the unit on. The on/off switch is diagrammed in your manual. It will count down 600 seconds to warm up.

4.2.2 After the instrument warms up, insert the cuvette holder into the sample chamber. Note that the top of the cuvette holder has an arrow shape molded to the top part of the holder. Orient this arrow pointing toward the silver alignment mark on the inside rim of the sample chamber.

4.23 You will be performing a simple mode calibration (refer to your manual if need be). Press [enter] from the keypad. Enter [1] on the keypad to enter "setup", then enter [1] to enter "mode". Using the [arrow] key to choose the mode, select "simple", then enter [ESC] twice to return to the setup/calibration screen. Enter [2], then press [enter]. The TD-700 will prompt you to insert a typical sample. Place the test tube with your standard GFP solution inside the chamber, close the lid, and press [enter]. The TD-700 will now automatically set the standard to read 500 out of a total reading of 1,000 (the standard is set to 50% of maximum). When the screen indicates that the sample is 500, press [enter]. Your TD-700 is now calibrated.

5. Quantitating GFP

5.1 Place the sample to be assayed for GFP into a 13 x 100 mm borosilicate test tube. Make sure that the volume in the tube is over 3 ml. Anything under this volume will not clear the slits on the cuvette holder and the meniscus of the sample will interfere with excitation and emission.

5.2 Place the tube into the chamber and close the lid. The unit will measure the sample instantly.

5.3 Wait approximately 20 seconds for the signal to stabilize before recording it. If the output is 1,000 or over, the unit will read "OVER" indicating that the sample is too concentrated to read at the current sensitivity level. If this occurs, it is necessary to dilute your sample and perform another reading.

Note: The signals on fluorometers tend to drift over time, always insert your standard and record its value, (which is never far from 500) before taking any reading.

6. Example Quantitation

You have a strain of yeast expressing GFP under the sole control of a promoter used to normally control an enzyme that detoxifies a carcinogen. You grow a 10 liter culture of this strain to a predefined cell density, expose it for a period of time to a known concentration of this carcinogen, collect the cells, and prepare a cell extract. This extract is at a final volume of 1 liter. You prepare a 1 to 4 dilution of this extract by diluting 1 ml of the extract to a final volume of 4 ml with 20 mM Tris at pH 8.00. You mix this and derive a reading of 663 on your TD-700. Your standard was reading 510 at the time of the reading. You perform the following calculation to determine the amount of GFP induced in that exposure period for that amount of cells.

[(sample reading x dilution)/standard reading]
x 0.005 mg/ml x volume of extract

in this case
[(663 x 4)/510] x 0.005 mg/ml x 1000 ml

= 26 mg of GFP produced during the period of exposure in 10 liters of yeast culture.

REFERENCES

1. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C., © 1994, Green Fluorescent Protein as a Marker for Gene Expression. Science. 263:802-805.
2. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J., © 1992, Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 111:229-233.
3. Inoue, S., and Tsuji, F.I. © 1994, Aequorea green-fluorescent protein: Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Letters. 341:277-280.
4. Wang, S., and Hazelrigg, T. © 1994, Implications for bed mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature. 369:400-403.
5. Shimomura, O., Johnson, F.H., and Saiga, Y., © 1962, Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. and Comp. Physiol. 62:1-8.
6. Morin, J.G., and Hastings, J.W., © 1971, Energy Transfer in a Bioluminescent System. J. Cell Physiol. 77:313-317.
7. Ward, W.W., Cody, C.W., Hart, R.C., and Cormier, M.J., © 1980, Spectrophotometric identity of the energy-transfer chromophores in Renilla and Aequorea green-fluorescent proteins. Photochem. Photobiol. 31:611-615.
8. Cody, C.W., Prasher, D.C., Westler, W.M., Prendergast, F.G., and Ward, W.W., © 1993, Chemical Structure of the Hexapeptide Chromophore of the Aequorea Green-Fluorescent Protein. Biochemistry. 32(5):1212-1218.

This GFP application note was written by Daniel G. Gonzalez M.S., who is currently a Biochemistry Ph.D candidate working in the laboratory of William W. Ward at Rutgers University (New Brunswick, N.J.). His interests include the physical characterization of chromophore formation in a variety of Green-Fluorescent proteins from various organisms. William W. Ward is one of the pioneers in GFP research and is still very active in the field. Both he and Daniel currently use a wide assortment of techniques in fluorescence analysis, protein purification and molecular biology to study and characterize Green-Fluorescent proteins.

The Ward lab currently prepares and coordinates a series of short courses in biotechnology that features Aequorea GFP as a model protein for purification and molecular manipulation. For information on these courses, contact Daniel at: Phone: (908) 932-9071, ext. 212, e-mail: meton@rci.rutgers.edu, Mailing address: Daniel Gonzalez, Rutgers University, Cook College, Biochemistry and Microbiology, New Brunswick, NJ 08903-0231.

Daniel wishes to thank Turner BioSystems for making this applications note possible and for providing the GFP community with a useful analysis tool in their TD-700 fluorometer.