This Small Business Innovation Research Phase I project focuses on development of an in vivo assay for the family of Interleukin 1(beta) converting enzyme (ICE) protease, activate the apoptotic pathway is not clear, and progress in this area has been limited due to the lack of a reliable system to quantify these proteases. The proposed assay utilizes technology based on the green fluorescent protein (GFP), a naturally fluorescent protein which does not require additional factors to fluoresce. We propose to engineer an ICE-like protease cleavage sit unto GFP, thereby creating a naturally fluorescent substrate for these proteases. Fluorescence from GFP will be abolished upon cleavage at this site, providing a simple means to quantify protease activity. The assay is noninvasive, and allows researchers to quantify protease activity in real time on a single cell basis. PROPOSED COMMERCIAL APPLICATION: We plan to utilize the vectors and cell lines produced in Phase I to produce kits for the research market. Moreover, the assay will provide a useful model for the development of additional protease assays by insertion ofspecific cleavage sites into GFP. IN addition, the assay will facilitate identificaiton of factors which modulates ICE-like protease activity, and may facilitate therapeutic strategies for treatment of apoptosis related diseases.
Thesaurus Terms:bioassay, endopeptidase, enzyme activity, method development, programmed cell death, protein engineering active site, enzyme substrate, green fluorescent protein, interleukin 1 cell line, flow cytometry, fluorescence microscopy, polymerase chain reaction, transfection vectorNational Institute of General Medical Sciences (NIGMS)
