The overall goals of the research proposed are to develop a new generation of highly sensitive and stable biochemical labels based on the recombinant bioluminescent proteins aequorin and Renilla luciferase, which have recently been cloned and overproduced in our laboratory. Typical biochemical detection methods, such as radioimmunoassays and enzyme-linked immunosorbant assays, use radioisotopes and enzymes, respectively, as labels. Although these labels have demonstrated utility, they are limited in their general applicability because of problems such as disposal of radioisotopes, frequent lack of desired sensitivity, inhibition by plasma components, and difficulty in long-term storage and stability. A class of potential labels which could overcome many of these drawbacks are the bioluminescent proteins. Our laboratory has recently cloned and expressed two recombinant bioluminescent proteins, aequorin (from Aequorea victoria) and Renilla luciferase. Preliminary results indicate that these proteins are stable, derivatizable, and detectable in the range of 10-19 mol using commercially available photometers. Both proteins utilize coelenterate luciferin as a substrate. Aequorin (produced as a recombinant apoaequorin regenerated to aequorin in the presence of luciferin) produces a flash of light in response to Ca+2. In contrast, Renilla luciferase, which is entirely unlike the firefly luciferase, produce! a more sustained production of light by its simple catalytic action on coelenterate luciferin.
The specific aims of the proposed investigation are the following.
Aim #1 A variety of biotinylated derivatives of both apoaequorin, aequorin and luciferase will be prepared and tested for stability, sensitivity to detection, and affinity to avidin.
Aim #2 Apoaequorin, aequorin and luciferase will be covalently coupled to streptavidin and/or avidin to prepared conjugates. These conjugates will be tested for their stability, sensitivity to detection and affinity to biotin.
Aim #3 Apoaequorin, aequorin, and luciferase will be covalently coupled to goat-antimouse IgG and IgM and the derivatives will be tested for their stability, sensitivity to detection and affinity to antigens.
Aim #4 The derivatives prepared above for both aequorin and luciferase will be tested directly for their utility in solid-phase ELISA-type assays by head-to-head comparison against horseradish peroxidase and alkaline phosphatase. As a model system for these immunoassays the Forssman glycolipid and mouse monoclonal antibodies to it will be used. The results of these studies should make available a new generation of enzyme labels that could dramatically enhance the sensitivity and speed of diagnostic assays for cancer and other diseases and facilitate early detection and monitoring of therapeutic procedures.
| Ehrig, T; O'Kane, D J; Prendergast, F G (1995) Green-fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett 367:163-6 |
| Stults, N L; Stocks, N F; Rivera, H et al. (1992) Use of recombinant biotinylated aequorin in microtiter and membrane-based assays: purification of recombinant apoaequorin from Escherichia coli. Biochemistry 31:1433-42 |
