Aequorin variants with improved bioluminescence properties.

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs.

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

Whole-cell biosensing of 3-chlorocatechol in liquids and soils.

A rapid and sensitive technique is needed to analyze water and soils for chlorocatechols, common environmental pollutants produced from wood pulp chlorination and other processes. The soil bacteria Pseudomonas putida, harboring plasmid pSMM50R-B', selectively express beta-galactosidase in response to 3-chlorocatechol in pure water samples. The objective of the study was to determine whether background matrices in fresh water, sea water, soils, and organic solvents interfered with 3-chlorocatechol analysis by use of a bacteria-sensing system and by high-performance liquid chromatography (HPLC). Although 3-chlorocatechol detection by HPLC was not substantially affected by the background composition of aqueous or organic solvents, HPLC was ineffective in the analysis of contaminated soils due to irreversible contaminant sorption. Whereas detection by the bacteria-sensing system was reduced in the presence of aqueous and organic solvents, interferences could be reduced by sample dilution. 3-Chlorocatechol was detected when the bacteria were added directly to contaminated soils, suggesting that the organism enhanced desorption or had access to the sorbed compounds. Results indicate that the bacteria-sensing system has wide application for detection of 3-chlorocatechols in environmental samples, especially in soils where extraction and HPLC analysis are not efficient due to extensive contaminant sorption.

Bioluminescence immunoassay for thyroxine employing genetically engineered mutant aequorins containing unique cysteine residues.

Genetically engineered one-to-one conjugates between an analyte and a protein label have been demonstrated to yield assays with better detection limits and performance characteristics than those prepared by conventional chemical conjugation methods. To date, the preparation of these conjugates has been limited to fusion techniques where a peptide analyte is fused in frame to the protein label. To further expand the range of analytes that can be detected by using genetic engineering techniques coupled with bioanalytical methods, we have employed site-directed mutagenesis to prepare one-to-one analyte-label conjugates that include nonpeptidic analytes such as drugs, vitamins, and hormones. Specifically, we have prepared mutants of the photoprotein aequorin containing single cysteine residues suitable for site-specific conjugation. Aequorin is a photoprotein that emits light at 469 nm and has been employed as a highly sensitive bioluminescent label in the development of binding assays for important biomolecules. We have performed polymerase chain reaction-based site-directed mutagenesis on apoaequorin to yield four mutant aequorins containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain for the purpose of site-specific conjugation to a model analyte. A maleimide-activated thyroxine was selected as the model analyte and site-specifically conjugated to the mutants through their unique cysteine residues. A heterogeneous assay for thyroxine was then developed by employing the genetically engineered aequorin mutants.

Using epitope-aequorin conjugate recognition in immunoassays for complex proteins.

Recombinant techniques are used to fuse biologically important molecules or peptides to the N-terminus of the photoprotein aequorin such that the binding characteristics of the molecule and the bioluminescent activity of aequorin are retained. This work demonstrates that the peptide region of a bulky protein can be used to develop an assay for the protein. A heterogeneous competitive binding assay was first developed for HPC4 epitope, the binding region of protein C, using HPC4-apoaequorin conjugate. It was observed that the binding of HPC4 epitope to its monoclonal antibody and the bioluminescence properties of aequorin were retained in the fusion protein. The same strategy and the same fusion protein were used to develop the assay for protein C. This project could potentially be a model for large biomolecules utilizing only the binding region of the protein in the labeled analyte. Also, this assay can be used in clinical diagnostics for the quantitative detection of protein C.

Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction. Optimization and application to single cell analysis.

A class-selective post-capillary reaction detection method for capillary electrophoresis is described in which a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) conjugate is used to detect biotin moieties. The selective binding of biotin moieties to the streptavidin-FITC conjugate causes an enhancement of fluorescence proportional to the concentration of biotin present. After capillary electrophoresis the separated analytes react with streptavidin-FITC in a coaxial reactor and are then detected either by a benchtop spectrofluorometer (2.5 microM detection limit) or by an epi-fluorescence microscope (1 x 10(-7) M detection limit). The method is used to examine biotinylated species in a crude mammalian cell lysate which was found to contain 83+/-3 fmol in 3600 cell volumes. In addition, it is used to examine the uptake of biotin by individual sea urchin oocytes. The results indicate that, in the oocytes, biocytin is the prevalent form of biotin and its concentration varies widely between cells (mean=2+/-2 microM).

A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein.

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.

An immunoassay for Leu-enkephalin based on a C-terminal aequorin-peptide fusion.

Recently we demonstrated that the fusion of an octapeptide to the C-terminus of a cysteine-free mutant of aequorin showed no inhibitory effect on the luminescence activity of the photoprotein. This observation is of particular importance when the use of aequorin as a label in the development of immunoassays for peptides whose activity lies in their C-terminal region or the epitope for antibody recognition is at their C-terminus is desired. In the case of opioid peptides, antibodies are directed toward their C-terminus as they differ from each other at this terminus. The goal of this study was to develop an immunoassay for Leu-enkephalin, a mammalian opioid peptide, using a C-terminal aequorin-peptide fusion protein. For that, the N-terminus of Leu-enkephalin was genetically fused to the C-terminus of a cysteine-free mutant of aequorin. It was observed that the C-terminal conjugated aequorin maintained its luminescence activity. An immunoassay for Leu-enkephalin was then developed using the aequorin-Leu-enkephalin fusion protein as a labeled analyte in a competitive as well as in a sequential binding mode. It was demonstrated that aequorin can be used as a label in peptide assays in which it is critical that the peptide's C-terminus be free for activity and/or for antibody recognition.

C-terminal and n-terminal fusions of aequorin with small peptides in immunoassay development.

Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis.

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

Green fluorescent protein mutant as label in homogeneous assays for biomolecules.

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.

Chlorocatechol detection based on a clc operon/reporter gene system.

A sensitive and selective sensing system for chlorocatechols (3-chlorocatechol and 4-chlorocatechol) was developed based on Pseudomonas putida bacteria harboring the plasmid pSMM50R-B'. In this plasmid, the regulatory protein of the clc operon, ClcR, controls the expression of the reporter enzyme beta-galactosidase. When bacteria containing components of the clc operon are grown in the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream from the beta-galactosidase gene. Thus, the concentration of chlorocatechols can be related to the production of beta-galactosidase in the bacteria. The concentration of beta-galactosidase expressed in the bacteria was determined by measuring the chemiluminescence signal emitted with the use of a 1,2-dioxetane substrate. ClcR has a high specificity for chlorocatechols and provides the sensing system with high selectivity. This was demonstrated by evaluating several structurally related organic compounds as potential interfering agents. Both 3-chlorocatechol and 4-chlorocatechol can be detected with this sensing system at concentrations as low as 8 x 10(-10) and 2 x 10(-9) M, respectively, using a 2-h induction period. In the case of 3-chlorocatechol, a highly selective sensing system was developed that can detect this species at concentrations as low as 6 x 10(-8) M after a 5-min induction period; the presence of 4-chlorocatechol at concentrations as high as 2 x 10(-4) M did not interfere with this system.

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin.

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.

Electrochemistry in nanovials fabricated by combining screen printing and laser micromachining

The coupling of screen-printing and laser micromachining technology has been used to create a nanovial with "built-in" working and reference electrodes. The volume of the nanovial was calculated to be 7.2 nL using dimensions determined by SEM. The electrochemical nanovial was characterized using the ferri/ferrocyanide redox couple. Cyclic voltammetry and chronoamperometry experiments were performed with electrochemical nanovials utilizing 5% (v/v) glycerin in the solutions and a humidified headspace to control evaporation of the small-volume samples. Chronoamperometry experiments gave results consistent with a diffusion-limited process and revealed a working electrode surface area of 2.6 x 10(4) micron 2. The ultrasmall-volume cells represent a simple, reliable, low-cost approach for the fabrication of complete electrochemical nanovials.

Site-specifically labeled photoprotein-thyroxine conjugates using aequorin mutants containing unique cysteine residues: applications for binding assays (Part II).

The jellyfish Aequorea victoria produces a protein, aequorin, which belongs to the class of Ca(2+)-dependent photoproteins known for their ability to emit visible light. This property of aequorin has allowed for its as a bioluminescent label in binding assays for a variety of analytes. Due to the excellent detection limits we demonstrated in assays for small peptides using a fusion protein between the peptide of interest and the photoprotein, our next goal was to expand the range of possible analytes for producing homogeneous populations of conjugates with the aequorin label to those that were nonpeptidic in nature. Recently, we prepared and characterized four aequorin mutants containing unique cysteine residues at various positions in the polypeptide chain. In the work reported here, the four aequorin mutants were each conjugated with a maleimide-activated methyl ester derivative of thyroxine, a hormone frequently determined to evaluate thyroid function. The thyroxine-aequorin mutant conjugates were characterized in terms of the bioluminescence activities and binding properties with an anti-thyroxine monoclonal antibody for possible future employment in either heterogeneous or homogeneous binding assays for thyroxine and/or other desired analytes.

Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization.

Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca(2+)-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO(2) and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.

Photoproteins as luminescent labels in binding assays.

Certain marine organisms produce calcium-activated photoproteins that allow them to emit light for a variety of purposes, such as defense, feeding, breeding, etc. Even though there are many bioluminescent organisms in nature, only a few photoproteins have been isolated and characterized. The mechanism of emission of light in the blue region is the result of an internal chemical reaction. Because there is no need for excitation through external irradiation for the emission of bioluminescence, the signal produced has virtually no background. This allows for the detection of the proteins at extremely low levels, making these photoproteins attractive labels for analytical applications. In that regard, the use of certain photoproteins, namely, aequorin, obelin, and the green fluorescent protein as labels in the design and development of binding assays for biomolecules has been reviewed. In addition, a related fluorescent photoprotein, the green fluorescent protein (GFP), has been recently employed in bioanalysis. The use of GFP in binding assays is also discussed in this review.