Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin

Detection of proteolytic bond cleavage was achieved by taking advantage of the bioluminescence emission generated by the photoprotein aequorin. A gene tically engineered HIV-1 protease substrate was coupled with a cysteine-fre e mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage sit e. The fusion protein was immobilized on a solid phase and employed as the substrate for the HIV-1 protease. Proteolytic bond cleavage was detected by a decrease in the bioluminescence generated by the aequorin fusion protein on the solid phase. A dose-response curve for HIV-1 protease was construct ed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and o ne noncompetitive inhibitor of the HIV-1 protease. Among the advantages of this assay is that by using recombinant methods a complete bioluminescently labeled protease recognition site can be designed and produced. The assay yields very sensitive detection limits, which are inherent to bioluminescen ce-based methods. An application of this system may be in the high-throughp ut screening of biopharmaceutical drugs that are potential inhibitors of a target protease. (C) 2000 Academic Press.