Report
Applications of Reporter Genes
R
eporter genes code for proteins that produce a signal that allows the protein to be determined in a complex mixture of other proteins and enzymes. Thefirstuses for reporter genes were in studies of transcriptional activity in cells and have led to a greater understanding of the mechanisms of genetic regulation. Reporter genes are now being used as part of a signal transduction event in many biosensing systems and have been used to develop assays for such diverse compounds as metal ions, toxic organic species, viruses, and antibodies. In many analytical systems, a molecular recognition event is coupled to a reporter event to provide the required selectivity and sensitivity to detect an analyte. The molecular recognition event determines the selectivity of the system, whereas the reporter event generates a signal and, thus, ultimately controls the method's sensitivity. Recombinant DNA technology can be used to design the recognition and reporter processes at the molecular level. The ability to alter a specific property or properties of a protein offers unique opportunities for developing analytical methods. For example, a protein can be engineered to have a specific orientation when it is immobilized on a solid phase to improve its catalytic activity and stability (1). In addition, wellldefined conjugates between an analyte and a desired protein for assay development can be produced in which the protein functions as a label (2), and gene fusion approaches can be used to fuse an affinity tag to proteins to facilitate bioaffinity separations (3). An important advantage of
Jennifer C. Lewis Agatha Feltus C. Mark Ensor Sridhar Ramanathan Sylvia Daunert University of Kentucky
Reporter proteins can be used in bioanalytical methods to produce signalsindicatingthe presence of a target analyte. using recombinant proteins is the minimization of lot-to-lot variations in nrotein nreparation because once the gene eo the erotein is isolated and cloned, the recombinant protein can be reproduced at any time. This Report discusses emerging bioanalytical methods that take advantage of reporter genes to produce a signal that can be related to the presence of a target ana-
lyte. Reporter genes can express a reporter protein or they can be genetically manipulated to express a fusion protein of the reporter protein with another protein of interest. The latter strategy, for example, is used in cell trafficking studies (4). In that respect, reporter proteins can be used to produce signals for monitoring many cellular properties and events.
Analytical Chemistry News & Features, September 1, 1998 579 A
Report Reporter proteins
Left to right: Bacteria containing plasmid with cobA Q6ne, blue flourescent protein, GFP.
The availability of a wide variety of naturally occurring and genetically engineered reporter genes allows an extensive number of reporter proteins to be monitored by a variety of detection systems, such as electrochemical, fluorescence, and bio- and chemiluminescence. Researchers also have at their disposal, among the several luminescence-based reporter gene systems, a wide range of reporter proteins emitting at different wavelengths. Thus, there exists a palette of colors (photo above) to choose from, depending on the application. Additional reporter proteins with different emission wavelengths are continuously becoming available, further extending the range of applicability of these methods. For example, many different organisms, including single-celled algae, sea walnuts, jellyfish, fireflies, worms, and even some mushrooms are bioluminescent because certain photoproteins or enzymes are present. Hence, all these organisms could be a source of reporter proteins.
Desirable characteristics in a reporter protein include detection with high sensitivity, wide dynamic range of response, and ease of use. Moreover, the ideal reporter protein should be environmentally safe, giving rise to an easily discernible signal from the background. Sensitivity can be a function of several factors, including the detection method, efficiency of expression, reporter protein turnover number (if the reporter is an enzyme), and, if applicable, the endogenous levels of the reporter protein. One of the first proteins to be used as a reporter was chloramphenicol acetyltransferase (CAT) from E. colliTable e). This protein, like other nonluminescent reporter proteins, uses synthetic substrates that generate products that can be monitored by different detection systems (5). CAT catalyzes the transfer of the acetyl group from acetylcoenzyme A (acetyl-CoA) to the antimicrobial drug chloramphenicol. The system originally developed used 14C-labeled chloramphenicol or 14C-labeled acetyl-CoAto form a radioactive product that was monitored after extraction. It is now possible to use fluorescent chloramphenicol substrates which, upon action of CAT, form the corresponding acetylatedfluorescentproduct. It is still necessary, however, to separate the
Table 1 . Reporter proteins. Reporter protein
Reaction
Detection
Chloramphenicol acetyltransferase Alkaline phosphatase p-Galactosidase p-Glucuronidase Bacterial luciferase
Acetylation of chloramphenicol or its derivatives
Rl, FL
Phosphate hydrolysis Hydrolysis of p-galactosides Hydrolysis of (3-glucuronides FMNH2 + decanal + 0 2 -> FMN + decanoic acid + H 2 0 + hv (490 nm) ATP + firefly luciferin + 0 2 -> AMP + oxyluciferin + inorganic phosphate + hv (560 nm) Apoaequorin + coelenterazine + 0 2 + Ca 2+ —> apoaequorin + coelenteramide + C0 2 + hv (469 nm) Apoobelin + coelenterazine + 0 2 + Ca 2+ —> apoobelin + coelenteramide + C0 2 + hv (470 nm) Posttranslational formation of an internal chromophore Transmethylation of urogen III to precorrin-2; precorrin-2 oxidized to sirohydrochlorin and is transmethylated to trimethylpyrrocorphin
EC, CL, FL EC, CL, FL EC, CL, FL BL
Firefly luciferase Aequorin
Obelin
Green fluorescent protein Urogen III methyltransferase
BL BL
BL
FL FL
BL, bioluminescence; CL, chemiluminescence; EC, electrochemical; FL, fluorescence; Rl, radioisotope.
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Analytical Chemistry News & Features, September 1, 1998
product from the substrate. This poses a disadvantage in CAT-based methods. The reason CAT is still used as a reporter protein is that its activities are not native to eukaryotic cells. Alkaline phosphatase, p-galactosidase, and p-glucuronidase are well-studied enzymes that have been used as reporter proteins. Numerous alkaline phosphatases are of both bacterial and mammalian origin. The common feature of these enzymes is that they are phosphohydrolases that function optimally at alkaline pH. A drawback is that some form of alkaline phosphatase activity is present in practically all cell types. On the other hand alkaline phospti3.t3.se s 3i*e versatile because a wide be detected with different methods depending on the desired aDolication (Table 1) (6) Bacterial p-galactosidase encoded by the gene lacZotE. coli catalyzes the hydrolysis of p-galactosides (Table 1). The P-galactosidase gene can be successfully used in prokaryotic and eukaryotic cells. The level of endogenous P-galactosidase activity varies greatly within cells; however, it is possible to distinguish between mammalian and bacterial enzymatic activity by altering the pH. p-glucuronidase is one of the most popular reporter proteins for use in plants. Many plants lack endogenous p-glucuronidase activity (7). This enzyme is a hydrolases catalyzing the cleavage of p-glucuronides (Table 1). Alkaline phosphatase, P-galactosidase, and p-glucuronidase are all enzymes that have a high turnover number and can generate a strong signal using any one of a number of fluorescence, electrochemical, and chemiluminescence substrates, many of which are sold as part of commercially available kits. The use of luminescent reporter proteins has grown in recent years because of the proteins' low detection limits and low, ,f any, endogenous activity in most cells. The most commonly used luminescent reporter proteins include luciferase (bacterial and firefly), aequorin, and the green fluorescent protein (GFP) (8). The genes coding for each of these proteins have been isolated. Other luminescent proteins exist, and research continues to discover new light-emitting proteins and their corresponding genes. Efforts
Figure 1 . Regulation of a reporter gene by a regulatory protein. Binding of the regulatory protein R to the promoter P controls transcription, followed by translation of the mRNA to produce the protein. Both of these steps produce multiple copies of the reporter protein, leading to an increased protein concentration.
also continue to modify known genes to aller excitation and emission wavelengths, increase luminescence quantum yields, and enhance stabilities. Bacterial luciferases and the genes that code for them have been isolated from numerous marine, freshwater, and terrestrial bacteria. Although the amino acid and nucleotide sequences of these luciferases differ, they catalyze the same bioluminescent reaction. Bacterial luciferases are flavin-dependent monooxygenases that catalyze the oxidation of reduced flavin mononucleotide (FMN) and a long-chain aldehyde to FMN and the corresponding long-chain carboxylic acid with light emission at 490 nm The quantum yield of this reaction is —0 1 The different strains of bacteria that produce the luciferases have sets of genes that responsible for the bioluminescence properties of these microorganisms The penes luxA and luxB code
cence response than does tetradecanal. It is possible to use the luxA and luxB genes in reporter systems without the other lux genes. The luciferase activity is then determined by adding decanal to the system exogenously (9). Firefly luciferase was first isolated from the North American firefly Photinus pyralis and differs from the bacterial luciferases in its structure and light-emitting reaction. Firefly luciferase is encoded by the luc gene. In the presence of adenosine triphosphate (ATP) and molecular oxygen, firefly luciferase catalyzes the oxidation of its substrate luciferin to oxyluciferin yielding C0 2 and light (Ji^ = 560 nm, quantum yield = 0.88). The bioluminescence signal is linear over 8 orders of magnitude of luciferase concentration and the enzyme can be detected at subattomole levels (10) making this luciferase an attractive choice for quantitative analytical applications
for the a and pt snhunits of harterial InHf-
The Ca2+-dependent photoprotein aequorin was first isolated from the jellyfish Aequorea victoria, found in Puget Sounund Washington state. The protein is composed of the apoprotein apoaequorin and an imidazopyrazine chromophore, coelenterazine. The binding of Ca2+ to aequorin induces a conformational change in the pro-
and genes luxF rode for enzymes respon'sible for svnthesis of the lontr-chain aldehyde snhtr t Alth rrh tetradecanal is the natural
substrate synthesized by luciferase-containing bacteria, shorter chain aldehydes (e.g., decanal) elicit a much higher lumines-
tein, triggering the oxidation of the coelenterazine to coelenteramide with the release of C0 2 and a flash of light at 469 nm (quantum yield = 0.15) (8). The protein can be detected at subattomole levels, which makes aequorin well-suited as a label in immunoassays (11,12), in hybridization assays (13), and as an indicator for measuring intracellular calcium in both prokaryotic and eukaryotic cells (8) like aequorin, the GFP was originally isolated from the jellyfish Aequorea victoria. Following the cloning of the GFP gene (14), the protein has been used extensively as an in vivo marker and as a monitor of dynamic processes inside living organisms (4). Native GFP has excitation maxima at 395 nm and 470 nm and emits light at 509 nm with a shoulder at 540 nm (see photo on p. 580 A). The quantum yield of GFP (= 0.85) is comparable with that of fluorescein. The fluorescence of GFP is the result of an internal chromophore formed by the autocatalytic posttranslational cvclization of three amino acids Ser65-Tvr66Gly67 (IS) The unique three-dimensional structure of GFP a series of 11 B-sheets internal chromophore the surroundine- environment (16) GFP has several characteristics that make it an excellent reporter protein. Unlike other bioluminescent proteins and enzymes, GFP does not require substrates or cofactors to emit light. Because of the protected location of the chromophore inside the P-barrel of the protein, GFP retains its fluorescence capability under mild denaturants, heat, detergents, and most common proteases. The fluorescence is also stable at pH 7-12 (14). In addition, GFP is nonlethal when expressed at high levels in various cells Most important mutants of the protein with different spectral properties be created such as increased fluoresintensity and shifted wavelengths of excitation and emission For example the photo on o 580 A shows a variant of GFP that emits blue fluorescence Uroporphyrinogen III (urogen III) is an intermediate in the biosynthetic pathways for heme, vitamin B12, and chlorophyll. Uroporphyrinogen III methyltransferase is encoded by the cobA gene en Propionibacteriumfreudenreichiior the cysG gene in E. coli. .I catalyzes the transfer of two meehyl
Analytical Chemistry News & Features, September 1, 1998 581 A
Report groups to urogen III, yielding dihydrosirohydrochlorin, also known as precorrin-2 (17). Precorrin-2 can either undergo oxidation to yield afluorescentproduct, sirohydrochlorin, or the transferase can catalyze the transfer of a third methyl group to urogen III, producing afluorescenttrimethylpyrrocorphin. In either case, the products formed yield red to red-orange fluorescence when illuminated with UV light at 300 nm. Urogen III methyltransferase does not require adding a substrate because the substrate urogen III is present in all organisms. This reporter system has been used for selecting recombinant plasmids for biotechnology applications (18). Cell-based sensing systems
Sensing systems typically contain a component that selectively recognizes an analyte, coupled to a detector that monitors the interaction between the sensing element and the analyte. Using whole cells as the sensing element offers some unique advantages. Compared with isolated proteins, whole cells are often less susceptible to changes in environmental conditions such as pH and temperature and the presence of other solutes. Most important is that, unlike classical methods these cell-based sensing systems provide a measure of the bioavailability of a given analyte rather than its total concentration because the analyte must be transDOrted inside the cells
able Microtox test was developed based on this principle, using the bioluminescent bacterium Vibriofischeri.The ttst predicts that the order of increasing metal ion toxicity is chromate ion
