Aequorin mutants with increased thermostability.

Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.

Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics.

Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.

Bioluminescence immunoassay for angiotensin II using aequorin as a label.

Angiotensin II is a biologically active component of the renin-angiotensin system. High levels of angiotensin II may be responsible for hypertension and heart failure because they increase systemic vascular resistance, arterial pressure, and sodium and fluid retention. Therefore, it is important to monitor angiotensin II levels for the treatment of hypertension and heart diseases. The goal of this work was to develop a bioluminescence immunoassay using aequorin as a label to measure angiotensin II levels in human plasma. This method utilizes a genetically engineered fusion protein between angiotensin II and aequorin. For that, the C terminus of angiotensin II was fused to the N terminus of apoaequorin using molecular biology techniques. A heterogeneous immunoassay was then developed for the determination of angiotensin II. A detection limit of 1 pg/mL was obtained with the optimized assay, allowing for the determination of angiotensin II at physiological levels in human plasma.