Red-emitting luciferases for bioluminescence reporter and imaging applications

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557 nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega’s codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.     

GroE-mediated folding of bacterial luciferases in vivo

In this study we present evidence indicating that GroE chaperonins mediate de novo protein folding of heterodimeric and monomeric luciferases under heat shock or sub-heat shock conditions in vivo. The effects of additional groESL and groEL genes on the bioluminescence of Escherichia coli cells expressing different bacterial luciferase genes at various temperatures were directly studied in cells growing in liquid culture. Data indicate that at 42° C GroESL chaperonins are required for the folding of the subunit polypeptide of the heterodimeric luciferase from the mesophilic bacterium Vibrio harveyi MAV (B392). In contrast, the small number of amino acid substitutions present in the luciferase subunit polypeptide from the thermotolerant V. harveyi CTP5 suppresses this requirement for GroE chaperonins, and greatly reduces interaction between the subunit polypeptide and GroEL chaperonin. In addition, GroESL are required for the de novo folding at 37° C of a MAV luciferase fusion polypeptide that is functional as a monomer. No such requirement for luciferase activity is observed at that temperature with a fusion of the CTP5 and subunit polypeptides, although GroE chaperonins can still mediate folding of the CTP5 fusion luciferase. Bacterial luciferases provide a unique system for direct observation of the effects of GroE chaperonins on protein folding and enzyme assembly in living cells. Furthermore, they offer a sensitive and simple assay system for the identification of polypeptide domains required for GroEL protein binding.     

An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega’s luc2 for reporter and imaging applications.     

Spatial structure of the novel light-sensitive photoprotein berovin from the ctenophore Beroe abyssicola in the Ca-loaded apoprotein conformation state

The bright bioluminescence of ctenophores, found in oceans worldwide, is determined by Ca^2 +-regulated photoproteins, functionally identical to and sharing many properties of hydromedusan photoproteins. In contrast, however, the ctenophore photoproteins are extremely sensitive to UV and visible light over the range of their absorption spectrum. The spatial structure of a novel light-sensitive photoprotein from the ctenophore Beroe abyssicola in its apoform bound with three calcium ions is determined at 2.0 Å. We demonstrate that the apoberovin is a slightly asymmetrical compact globular protein formed by two domains with a cavity in the center, which exactly retains the fold architecture characteristic of hydromedusan photoproteins despite their low amino acid sequence identity. However, the structural alignment of these two photoprotein classes clearly shows that despite the high similarity of shape and geometry of their coelenterazine-binding cavities, their interiors differ drastically. The key residues appearing to be crucial for stabilizing the 2-hydroperoxycoelenterazine and for formation of the emitter in hydromedusan photoproteins, are replaced in berovin by amino acid residues having completely different side chain properties. Evidently, these replacements must be responsible for the distinct properties of ctenophore photoproteins such as sensitivity to light or the fact that the formation of active photoprotein from apophotoprotein, coelenterazine, and oxygen is more effective at alkaline pH.     

Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence

Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.     

Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. Here, we present studies leading to the production of a set of red- and green-emitting luciferase mutants with bioluminescent properties suitable for expanding the use of the P. pyralis system to dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Using a combination of mutagenesis methods, we determined that the Ser284Thr mutation was sufficient to create an excellent red-emitting luciferase with a bioluminescence maximum of 615 nm, a narrow emission bandwidth, and favorable kinetic properties. Also, we developed a luciferase, containing the changes Val241Ile, Gly246Ala, and Phe250Ser, whose emission maximum was blue-shifted to 549 nm, providing a set of enzymes whose bioluminescence maxima were separated by 66 nm. Model studies demonstrated that in assays using a set of optical filters, the luciferases could be detected at the attomole level and seven orders of magnitude higher. In addition, in the presence of the Ser284Thr enzyme serving as a control, green light emission could be measured over a 10,000-fold range. The results presented here with the P. pyralis mutants provide evidence that simultaneous multiple analyte assay development is feasible with these novel proteins that require only a single substrate.     

Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. We present studies on the production of a set of thermostable red- and green-emitting luciferase mutants with bioluminescent properties suitable for dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Starting with the luciferase variant Ser284Thr, we introduced the mutations Thr214Ala, Ala215Leu, Ile232Ala, Phe295Leu, and Glu354Lys to produce a new red-emitting enzyme with a bioluminescence maximum of 610 nm, narrow emission bandwidth, favorable kinetic properties, and excellent thermostability at 37 degrees C. By adding the same five changes to luciferase mutant Val241Ile/Gly246Ala/Phe250Ser, we produced a protein with an emission maximum of 546 nm, providing a set of thermostable enzymes whose bioluminescence maxima were separated by 64 nm. Model studies established that the luciferases could be detected at the attomole level and six orders of magnitude higher. In microplate luminometer format, mixtures containing 1.0 fmol total luciferase were quantified from measurements of simultaneously emitted red and green light. The results presented here provide evidence that it is feasible to monitor two distinct activities at 37 degrees C with these novel thermostable proteins.     

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.     

Construction of a modified mini-Tn5 luxCDABE transposon for the development of bacterial biosensors for ecotoxicity testing

A mini-Tn5 transposon was modified to introduce a promoterless luxCDABE cassette from Vibrio fischeri into environmentally relevant bacterial strains in order to develop bioluminescence-based biosensors for toxicity testing. The mini-Tn5 luxCDABE transposon was chromosomally integrated downstream from an active promoter into two Pseudomonas strains (Pseudomonas fluorescens 8866 and Pseudomonas putida F1). Characterisation of the bioluminescent transconjugants demonstrated that the transposon integration was stable and had no effect on growth rate. Both P. fluorescens 8866 Tn5 luxCDABE and P. putida F1 Tn5 luxCDABE were used to assess the toxicity of standard solutions (Cu, Zn and 3,5-DCP) as well as Cu- and 3,5-DCP-spiked groundwater samples. They were successfully used for bioluminescence-based bioassays and the potential value of using different bacterial biosensors for ecotoxicity testing was shown.     

Isolation and characterization of Acidithiobacillus caldus from a sulfur-oxidizing bacterial biosensor and its role in detection of toxic chemicals

A novel toxicity detection methodology based on sulfur-oxidizing bacteria (SOB) has been developed for the rapid and reliable detection of toxic chemicals in water. The methodology exploits the ability of SOB to oxidize sulfur particles in the presence of oxygen to produce sulfuric acid. The reaction results in an increase in electrical conductivity (EC) and a decrease in pH. The assay is based on the inhibition of SOB in the presence of toxic chemicals by measuring changes in EC and pH. We found that SOB biosensor can detect toxic chemicals, such as heavy metals and CN−, in the 5-2000 ppb range. One bacterium was isolated from an SOB biosensor and the 16S rRNA gene of the bacterial strain has 99% and 96% sequence similarity to Acidithiobacillus sp. ORCS6 and Acidithiobacillus caldus DSM 8584, respectively. The isolate was identified as A. caldus SMK. The SOB biosensor is ideally suited for monitoring toxic chemicals in water having the advantages of high sensitivity and quick detection.     

Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.     

The Reliability of Marker/Reporter Genes in Biocontrol Studies with B. subtilis

A commercially available biocontrol agent, Bacillus subtilis MBI600, was transformed with plasmids containing a luciferase lux AB fusion cassette under the control of a constitutive promoter, as well as two promoter-less plasmids, containing either a lux AB fusion cassette or lux A and lux B genes. Introduction of these genes did not alter the growth rate, but subsequent expression of the luciferase enzyme resulted in a consistently compromised in vitro biocontrol activity. The expressed luciferase gene product (rather then the insertion of the novel genes) appeared to have undesired side effects on the phenotype of the transformed organism, demonstrating why caution should be exercised when using marker/reporter gene systems for investigating biocontrol.     

Controlled expression of click beetle luciferase using a bacterial operator-repressor system

Abstract The bioluminescent phenotype conferred by luciferase genes in a particular bacterium has demonstrated to be one of the most versatile and useful methods to detect microorganisms. Genetic constructions derived from miniTn5 vectors have been constructed for the introduction and stable maintenance of the click beetle luciferase gene, lucOR , in various Gram-negative bacteria. To attenuate the expression in the environment where the marked strain has to survive (and to allow sensitive detection when desired) a DNA fragment containing the repressor gene lacI ^q and a P _trc:: lucOR fusion was cloned onto a suicide plasmid. This construction is able to express high luciferase levels only when induced by IPTG. Matings between Escherichia coli containing the suicide transposoon vector and different recipient bacteria gave transposition frequencies from 10⁻⁷ to 10⁻⁵. Strains with miniTn5- lucOR insertions showed luciferase activity induced by IPTG addition. The stringency of the regulation and the intensity of light emission depended on the tagged strain. This system allows stable maintenance of the marker and tight control of luciferase expression in the environment.     

Factors affecting the cellular expression of bacterial luciferase

The in vivo expression of cellular bacterial luciferase has been defined as the luciferase expression quotient, measured as the ratio of the bioluminescence intensity in vivo to the in vitro activity of luciferase in crude cell extracts. The expression is greater in the presence of inhibitors of the electron transport system such as cyanide and N-heptyl-4-hydroxyquinoline and also at lower oxygen tensions. The higher expression of the cellular luciferase under these conditions is postulated to be due to an increase in the intracellular levels of reduced coenzymes which enhance both the reduction of flavin and the reduction of fatty acid to aldehyde. Both FMNH₂ and aldehyde are substrates in the light emitting reaction.     

Interaction of dimethyl-and monomethyloxyluciferin with recombinant wild-type and mutant firefly luciferases

Dissociation constants (Ks) in the pH range 6.5-9.0 for complexes of luciferin, dimethyloxyluciferin (DMOL), and monomethylluciferin (MMOL) with recombinant wild-type and mutant (His433Tyr) luciferases from the Luciola mingrelica firefly were determined by fluorescent titration. The protonated effectors were bound by the wild-type and mutant luciferases better than the nonprotonated ones. The affinity of DMOL for the mutant luciferase was higher than for the wild-type luciferase at alkaline pH, whereas the affinity of MMOL was higher at all pH values studied. The fluorescence emission and excitation spectra of DMOL and MMOL in buffer solution (pH 7.8) were obtained in the absence and presence of luciferase. The fluorescence maxima of DMOL and MMOL complexes with luciferase were 20 and 100 nm, respectively, shifted to shorter wavelengths as compared to the values in buffer solution. This was explained by nonspecific and specific influence of the protein microenvironment on the fluorescence spectra of DMOL and its specific influence on the MMOL fluorescence spectra.     

Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates.