The protein aequorin is a bioluminescent photoprotein, which emits light in the presence of calcium. This emitted light can be effectively detected using a microplate luminometer, and subsequently characterized using rapid, high throughput screening assays. Since various aequorin mutants have different emission properties, this process can be used to identify the mutant that provides the most desirable bioluminescence emission for the accurate and easy measurement of intracellular calcium concentrations.
Intracellular calcium ions play a vital role in a great deal of physiological processes; from the contraction of all muscle types, to fertilization and bone formation. As such, the accurate measurement of calcium levels can provide scientists with information about intracellular pathways, for example various signal transduction pathways. One way in which these calcium ion levels can be accurately measured is through the use of the photoprotein aequorin. Isolated from luminescent jellyfish and other marine organisms, aequorin is composed of two distinct units: the 22 kDa apoprotein apoaequorin; and the prosthetic group coelenterazine (CTZ). These two components will spontaneously reconstitute in the presence of molecular oxygen to form the functional protein.
Cultured cells expressing the aequorin gene have the ability to effectively express the aequorin protein. However, recombinant expression only yields the apoprotein. It is therefore necessary to add CTZ to the culture medium in order to obtain a functional protein. As a hydrophobic molecule, CTZ is able to pass easily across both plant cell walls as well as the more complex lipid bilayer of higher eukaryotes. Once inside the cell, it can interact with the apoaequorin to form an active protein. In its active state, the protein has several calcium ion binding sites, which once bound will result in a number of conformational changes. The CTZ is converted via an oxidation reaction to excited CTZ and carbon dioxide. As the excited CTZ returns to its ground state, it will emit light with a wavelength of 469 nm (in the blue part of the spectrum), which can be easily detected using a luminometer such as the Thermo Scientific Varioskan Flash. Kinetically, the reaction is very fast and it can often be problematic to accurately capture its activity.
This article will explore rapid, high-throughput screening assays of mutated Aequorin in order to find clones with the most intense, yet slower, narrow spectral or color-shifted bioluminescence emission kinetics. This screening requires special instrumentation to detect and quantify the activity of wild type and mutant aequorins expressed in the recombinant Escherichia coli (E.coli) cells. Due to its unique automatic dispensing features, the Varioskan Flash was used to detect the luminometric spectra. Due to the ability to perform various kinetic readings well-by-well, each measurement time is kept to approximately 10 ms.
Materials
All of the luminometric measurements were performed using the Varioskan Flash spectral scanning multimode microplate reader, controlled by the Thermo Scientific SkanIt Software. Random mutagenesis was performed using a plasmid for the expression of wild-type aequorin as a template. An E. coli strain was used as the bacterial host for expression and all assays were performed on a white 96-well microtitre strip plate.
Protocol
A CTZ stock solution was prepared at 1 mg/ml concentration in methanol acidified by hydrochloric acid (HCL). The CTZ was dissolved in amber microcentrifuge tubes to protect it from potentially harmful light. The working solution was prepared by diluting fresh CTZ to a final concentration of 5µM in a solution containing 100 nM Tris base, which has a pH of 8, 90 mM NaCl and 5 mM EDTA. The triggering solution was composed of 0.75% Triton X-100 and 15 mM CaCl2 in water.
Recombinant cells expressing aequorin were grown on selective LB broth to an optical density (OD600nm) of 0.6. Cells were subsequently transferred to a white 96-well microtitre plate in 150 µl aliquots and incubated with 50 µl CTZ-EDTA for five hours, in the dark, at 4 °C.
Since aequorin emission is released as a rapidly decaying flash upon calcium binding, the reaction ideally needs to be measured with an instrument that is equipped with an automatic dispenser. As such, the dispensing unit was primed with a triggering solution and set to dispense 10 µl at a high dispensing speed. The instrument temperature was set to ambient to maintain 23 °C.
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Figure 1: Luminescence kinetics of aequorin mutants (expressed in RLU, relative light unit) values |
Kinetic measurements and luminometric spectra
In order to accurately monitor the emission decay, the multimode reader was programmed to perform the following measurements:
Kinetic measurements
• Well loop step – the instrument was set to execution type ‘by well’, well count 1. This setting enables the execution of all actions to one well before proceeding to the next.
• Kinetic loop step – the instrument was set to perform 1000 readings with an interval of 0 seconds. This enabled the performance of a kinetic assay with 1000 kinetic points, performed rapidly with no intervals between readings.
• Dispensing step – dispenser 1 was set to dispense a volume of 10 µl at high speed. The dispensing position was set to luminometric 1, tip priming automatic with the dispense occurring at reading 10. This enabled the instrument to dispense 10 µl of Ca2+ triggering solution into each well.
• Measurement step – in order to obtain luminometric measurements, the luminometric optics were set to ‘normal’ with the dynamic range in the ‘autorange’ and a measurement time of 10 ms. This enables the reader to obtain kinetic measurements at a sampling speed of 100 points/second, since the total kinetic measurement time of the Aequorin signal is just 2.5 seconds.
Luminometric spectra
• Well loop step – execution type was set to ‘by well’, well count 1. This enabled the instrument to perform the following actions well by well.
• Dispensing step – Dispenser 1 was set to dispense 20 µl at a high speed. The dispensing position was luminometric 1 and tip priming was automatic.
• Measurement step – luminometric spectral scanning was set to a wavelength range of 400 – 600 nm and the dynamic range set to auto-range with a measurement time of 100 ms. This setting enabled the reader to measure the Aequorin spectra from the selected wavelengths in 2 nanometer steps.
The assay plate was positioned on the instruments plate carrier and the full program was executed. After all measurement steps were completed, the data was exported from the SkanIt software as a Microsoft Excel spreadsheet using the ‘Organized Export’ function. Data analysis was subsequently performed directly in Excel.
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Figure 2: Emission spectrum of wild-type aequorin |
Results
As seen in figure 1, aequorin light emission shows a maximum value at 469 nm and the luminescence peak has a bandwidth of approximately 80 nm. These results are analogous to previously reported values (Figure 2). This shows that the emission spectra of aequorin mutants can be accurately captured, and it is comparable to previously reported values.
Conclusion
The Thermo Scientific Varioskan Flash, which encompasses high-sensitivity luminometric capabilities with automatic dispensing and luminometric spectral scanning, is an ideal instrument to accurately screen a high number of aequorin clones which have been generated by random mutagenesis. The instrument’s automatic dispenser is essential to achieving reliable and reproducible kinetic data. Furthermore, the incorporation of the SkanIt software allows users to create and optimize the kinetic measurement protocols. With the ability to characterize the emission properties of aequorin mutants, the user can successfully identify clones with more intense, slower kinetic, narrow-spectral or color-shifted bioluminescent emission for the accurate measurement of intracellular Ca2+ levels.