This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (6) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Naqvi, T. Articles by Eglen, R. M. Search for Related Content PubMed PubMed Citation Articles by Naqvi, T. Articles by Eglen, R. M. Social Bookmarking                         What's this?

Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology

DiscoveRx Corp., 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA.

Anice Lim

DiscoveRx Corp., 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA.

Riaz Rouhani

DiscoveRx Corp., 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA.

Raj Singh

DiscoveRx Corp., 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA.

Richard M. Eglen

DiscoveRx Corp., 42501, Albrae Street, Suite 100, Fremont, CA 94538, USA.

The authors describe a homogeneous, high-throughput screening (HTS) assay for measuring protease activity and detection of inhibitors. The assay comprises a cyclic ß-galactosidase (ß-gal) enzyme donor peptide (ED) containing a protease-selective cleavage sequence. Alone, the cyclic peptide is inactive, but when linearized following protease cleavage, ED complements with ß-gal enzyme acceptor forming active ß-gal enzyme. This then catalyzes the formation of either fluorescent or chemiluminescent products, with ß-gal turnover providing a highly amplified signal, and thus an assay technology of high sensitivity. To demonstrate the utility of the technology, an EFC assay was developed to measure the activity of 2, caspase 3 and ß-secretase. Using a cyclic ED containing the caspase 3 substrate sequence, DEVD, the EFC assay signal was linear with respect to caspase 3 concentration. The assay was very sensitive, being able to detect activity at low picogram amounts of caspase 3. For the ß-secretase (BACE) EFC assay, a cyclic ED containing the Swedish mutant cleavage site of amyloid precursor protein (APP), SEVNLDAEFK, was used. In a similar fashion to the caspase 3 assay, the signal induced by BACE activity was linear with respect to enzyme concentration and was highly sensitive, being able to detect nanogram quantities of BACE. The assay was also more sensitive than a commercially available FRET-based assay of BACE activity. It is concluded that the EFC protease assay is a simple, flexible, and sensitive technology for HTS of proteases.

Key Words: enzyme fragment complementation • ß galactosidase • protease assays • caspase 3 • ß secretase • BACE

Journal of Biomolecular Screening, Vol. 9, No. 5, 398-408 (2004)
DOI: 10.1177/1087057104264040


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				C. Antczak, T. Takagi, C. N. Ramirez, C. Radu, and H. Djaballah Live-Cell Imaging of Caspase Activation for High-Content Screening J Biomol Screen, September 1, 2009; 14(8): 956 - 969. [Abstract] [PDF]

				H.-K. Lee, S. J. Brown, H. Rosen, and P. S. Tobias Application of beta-Lactamase Enzyme Complementation to the High-Throughput Screening of Toll-Like Receptor Signaling Inhibitors Mol. Pharmacol., October 1, 2007; 72(4): 868 - 875. [Abstract] [Full Text] [PDF]