| Two naturally occurring and recombinantly produced luminescent proteins, namely aequorin and the green fluorescent protein (GFP) have been employed to develop highly sensitive assays with applications in bioanalysis. These two proteins were originally isolated from the jellyfish Aequorea victoria found in the Pacific Northwest. In recent years, fluorescence and bioluminescence have become popular detection principles as they provide an excellent alternative to radiolabels due to their high sensitivity of detection and non-hazardous nature. In addition, we have employed genetic engineering tools to generate protein-analyte conjugates, which allows highly reproducible production of reagents in unlimited amounts.; An assay for detection of proteolytic bond cleavage and for the identification of protease inhibitors was developed using a fusion protein between a chosen recognition sequence for the HIV-1 protease and a mutant aequorin. This fusion protein was site-specifically immobilized on the 96-well microtiter plate. The assay was based on the change in bioluminescence signal obtained on the solid phase upon cleavage by the HIV-1 protease at the recognition site, thus releasing the aequorin label into the liquid phase.; Homogeneous assays are mix-and-measure type of assays that offer the advantage of reduced time and cost, hence are preferred over solid phase assays. Using a mutant of GFP (EGFP) as a label homogeneous assays for biotin and cortisol were developed by chemically conjugating the analyte to the protein. The assays were based on quenching of the fluorescence signal of the EGFP-analyte conjugate in the presence of the respective analyte binder.; In prior work, gene fusion to aequorin has been restricted only to its N-terminus, since it was reported that C-terminal modification leads to loss in bioluminescence activity of aequorin. In order to increase the flexibility of aequorin as a label, we have demonstrated that fusion can be performed through the C-terminus of aequorin by fusing two different peptides to its C-terminus. Immunoassays were developed for both peptides using the respective aequorin-peptide C-terminal fusion proteins. |
