Genetically encoded infrared fluorescent protease reporters
AssigneeThe Regents of the University of California
InventorsXiaokun Shu, Tsz-Leung To
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Abstract
The invention described herein features infrared fluorescent protease reporters (iProteases) and methods of use thereof. The iProteases can be used in in vivo and in vitro assays to detect protease activity and disease states associated with protease activity. In a still further embodiment, the present invention provides a kit comprising any of the above described polynucleotides. In a further aspect, the present invention provides a method of in vivo optical imaging. In a still further embodiment, the in vivo imaging is performed in a living animal. In a further aspect, the present invention provides a method of detecting protease activity, the method comprising expressing a polypeptide according to any of those described above in a cell.
Claims42
A variant polypeptide of a parent polypeptide, wherein said variant polypeptide comprises at least 90% identity to an amino acid sequence selected from the group comprising SEQ ID NOS:24-46.
An isolated polynucleotide encoding a polypeptide comprising at least 90% identity to a nucleic acid sequence selected from the group comprising SEQ ID NOS:1-23.
A method of making an infrared fluorescent protease reporter from an infrared protein (IFP) comprising:
- a. making a circular permutation to the IFP wherein the original N- and C-termini are linked together by a protease cleavage sequence,
- b. cutting the sequence between the PAS and GAF domains to create new N- and C-termini such that the C-terminus is on the PAS domain and the N-terminus is on the GAF domain,
- c. adding a split GFP to the new N- and C-termini,
- d. and truncating the original N- and C-termini so that the distance between the catalytic cysteine and the carboxyl end of the GAF is larger than the length of protease cleavage sequence.
A method of detecting apoptosis, the method comprising expressing an infrared fluorescent protease reporter that has an amino acid sequence of that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:40-42 in a cell, wherein the infrared fluorescent protease reporter fluoresces in response to the activation of apopotosis.
A method of detecting the efficacy of a therapy designed to induce apoptosis comprising:
- a. administering an infrared fluorescent protease reporter that has an amino acid sequence of that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS:40-42 to a subject;
- b. detecting the infrared fluorescence of the infrared fluorescent protease reporter,
- c. administering an apoptosis-inducing drug to the subject,
- d. detecting the infrared fluorescence of the infrared fluorescent protease reporter after the administration of the apopotosis-inducing drug,
- wherein an increase in infrared fluorescence is indicative of increased apopositis and the efficacy of the apoptosis-inducing drug.
A method of detecting calpain activity, the method comprising expressing an infrared fluorescent protease reporter that has an amino acid sequence of that is at least 90% identical to an amino acid sequence of SEQ ID NO:46 in a cell, wherein the infrared fluorescent protease reporter fluoresces in response to the activation of apopotosis.
A method of detecting the efficacy of a therapy designed to inhibit calpain activity comprising:
- a. administering an infrared fluorescent protease reporter that has an amino acid sequence of that is at least 90% identical to an amino acid sequence of SEQ ID NO:46;
- b. detecting the infrared fluorescence of the infrared fluorescent protease reporter,
- c. administering calpain inhibitor to the subject,
- d. detecting the infrared fluorescence of the infrared fluorescent protease reporter after the administration of the calpain inhibitor,
- wherein decrease in infrared fluorescence is indicative of decreased calpain activity and efficacy of the calpain inhibitor.
Dependencies are inferred from the claim text.