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Title:
N-END RULE PROTEASE ACTIVITY INDICATION METHODS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2012/047325
Kind Code:
A3
Abstract:
A cell based assay for detection for protease activity is disclosed. In the assay a cell is engineered to express a protease substrate with at least one label, preferably on its C-terminus. Cleavage of the substrate by the protease that recognizes it results in a C-terminal fragment and a N-terminal fragment, where the fragment having the label is subject to ubiquitin proteasome degradation. The assay measures the disappearance of the label due to degradation of the fragment to which it is attached. A cell free assay is also described for detection of protease activity. In the cell free assay, the protease substrate is expressed in a solution that includes the elements of the ubiquitin proteasome pathway for degradation of the fragment. The assay measures the disappearance of the label attached to the fragment that results from cleavage by the protease.

Inventors:
OYLER, George A. (2924 Guilford Avenue, Baltimore, Maryland, 21218, US)
TSAI, Yien Che (2538 Carrington Way, Frederick, Maryland, 21702, US)
Application Number:
US2011/040232
Publication Date:
July 26, 2012
Filing Date:
June 13, 2011
Export Citation:
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Assignee:
SYNAPTIC RESEARCH, LLC (1448 South Rolling Road, Baltimore, Maryland, 21227, US)
OYLER, George A. (2924 Guilford Avenue, Baltimore, Maryland, 21218, US)
TSAI, Yien Che (2538 Carrington Way, Frederick, Maryland, 21702, US)
International Classes:
C12Q1/37; C07K14/195; C12N15/57; C12N15/63; C12Q1/02; G01N33/68
Domestic Patent References:
WO2004031355A22004-04-15
Foreign References:
US20070122858A12007-05-31
Other References:
SUN S. ET AL.: "Multi-wavelength Spatial LED illumination based detector for in vitro detection of Botulinum Neurotoxin A Activity", SENS. ACTUATORS B CHEM., vol. 146, no. 1-8, 8 April 2010 (2010-04-08), pages 297 - 306, XP055070682
SUN S. ET AL.: "Lab-on-a-chip for botulinum neurotoxin a (BoNT-A) activity analysis", LAB ON A CHIP, vol. 9, no. 22, 21 November 2009 (2009-11-21), pages 3275 - 3281, XP055070684
SAPSFORD K.E. ET AL.: "A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity", BIOSENS. BIOELECTRON., vol. 24, no. 4, 1 December 2008 (2008-12-01), pages 618 - 625, XP025535127
TSAI Y.C. ET AL.: "Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system", PROC. NATL. ACAD. SCI. U. S. A., vol. 107, no. 38, 21 September 2010 (2010-09-21), pages 16554 - 16559, XP055070689
See also references of EP 2580345A4
Attorney, Agent or Firm:
MORALES, Joseph L. et al. (Whiteford, Taylor & Preston LLPSeven Saint Paul Stree, Baltimore Maryland, 21202-1636, US)
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