The photoprotein aequorin was originally isolated from jellyfish. Owing to its high sensitivity, low background and no toxicity, aequorin has recently widely used in biological and analytical applications, such as intracellular calcium detection, gene expression study, binding and immunoassay development, etc.
Recombinant aequorins can be easily obtained since the cDNA sequence of aequorin has been determined. Aequorin can be genetically conjugated to a target analyte to form a fusion protein which can be produced reproducibly and in a large amount. One immunoassay for angiotensin II detection using aequorin as a label has been developed. The proposed method, which utilized the aequorin-angiotensin II fusion protein, is simple and safe compared to conventional methods using radioisotopes, and is sensitive enough to estimate the angiotensin II levels in human plasma.
Aequorin and other calcium-binding photoproteins have similar bioluminescent properties such as similar maximum emission wavelengths and decay kinetics because all of them use coelenterazine as the substrate, which greatly limits its multiplexing capability (simultaneous detection of multiple analytes in a sample). To address this problem, two mutagenesis strategies, site-directed mutagenesis and random mutagenesis, coupled with different coelenterazine analogues, were used to get aequorin mutants that have improved bioluminescent properties.
First, several residues in the catalytic site of aequorin were selected for mutagenesis study by examining the X-ray crystal of the protein. The obtained mutants were characterized in terms of activity, half life, stability and spectral emission.
Next, error-prone PCR random mutagenesis was used to obtain several mutants with altered characteristics, especially two mutants with enhanced thermostability compared to wild type aequorin.
Finally, by combining a double mutation identified by our studies with another double mutation reported by another research group, a four-mutation mutant was constructed. The obtained mutant is much more thermostable than either of the two double mutants. Circular dichroism spectra of the four-mutation thermostable mutant were compared with those of wild type aequorin and the double mutant.
KEYWORDS: Photoprotein Aequorin, Bioluminescence Immunoassay Angiotensin II, Error-prone PCR, Mutagenesis