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 genetically engineered HIV-1 protease substrate was coupled with a cysteine-free mutant of aequorin by employing the polymerase chain reaction to produce a fusion protein that incorporates an optimum natural protease cleavage site. 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 constructed by relating the decrease in bioluminescence signal with varying amounts of the protease. The system was also used to evaluate two competitive and one 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 bioluminescence-based methods. An application of this system may be in the high-throughput screening of biopharmaceutical drugs that are potential inhibitors of a target protease.
Deo
S K
SK
Department of Chemistry, University of Kentucky, Lexington 40506, USA.
Lewis
J C
JC
Daunert
S
S
D000331:Aequorin / Q000378:metabolism*; D019921:Biotinylation; D004305:Dose-Response Relationship, Drug; D016333:HIV Protease / Q000187:drug effects / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D008027:Light; D008163:Luminescent Measurements; D010447:Peptide Hydrolases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D017320:HIV Protease Inhibitors; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D010447:Peptide Hydrolases; D016333:HIV Protease