Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin

Sapna K Deo, Jennifer C. Lewis, Sylvia Daunert

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)87-94
Number of pages8
JournalAnalytical Biochemistry
Volume281
Issue number1
DOIs
StatePublished - May 15 2000
Externally publishedYes

Fingerprint

Aequorin
Bioluminescence
Peptide Hydrolases
Fusion reactions
Assays
Luminescent Proteins
Immobilized Proteins
Preclinical Drug Evaluations
Proteins
Polymerase chain reaction
Cysteine
Limit of Detection
Screening
Throughput
Polymerase Chain Reaction
Pharmaceutical Preparations
HIV protease substrate 1
Human immunodeficiency virus 1 p16 protease

Keywords

  • Aequorin
  • Bioluminescence
  • Bond cleavage assay
  • HIV-1 protease
  • Protease inhibitors

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Bioluminescence detection of proteolytic bond cleavage by using recombinant aequorin. / Deo, Sapna K; Lewis, Jennifer C.; Daunert, Sylvia.

In: Analytical Biochemistry, Vol. 281, No. 1, 15.05.2000, p. 87-94.

Research output: Contribution to journalArticle

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