title: A bioluminescent assay for the sensitive detection of proteases.
abstract: A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the protein's stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.
authors.affiliation: Research and Development, Promega Corporation, Madison, WI, USA.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Leippe
authors.forename: Duy
authors.initials: D
authors.lastname: Nguyen
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Troy
authors.initials: T
authors.lastname: Good
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kirkland
authors.forename: Mike
authors.initials: M
authors.lastname: Scurria
authors.forename: Laurent
authors.initials: L
authors.lastname: Bernad
authors.forename: Tim
authors.initials: T
authors.lastname: Ugo
authors.forename: Jolanta
authors.initials: J
authors.lastname: Vidugiriene
authors.forename: James J
authors.initials: JJ
authors.lastname: Cali
authors.forename: Dieter H
authors.initials: DH
authors.lastname: Klaubert
authors.forename: Martha A
authors.initials: MA
authors.lastname: O'Brien
mesh_terms: D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D049454:Firefly Luciferin / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000378:metabolism*; D049408:Luminescent Agents / Q000737:chemistry; D008163:Luminescent Measurements / Q000379:methods*; D008956:Models, Chemical; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000032:analysis* / Q000145:classification / Q000378:metabolism; D011994:Recombinant Proteins / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D002364:Caseins; D005456:Fluorescent Dyes; D049408:Luminescent Agents; D009842:Oligopeptides; D011994:Recombinant Proteins; D049454:Firefly Luciferin; D049409:Luciferases, Firefly; D010447:Peptide Hydrolases