Mutant protease biosensors with enhanced detection characteristics

Patent metadata

Field	Value
Publication number	US-9290794-B2
Application number	US-201213674655-A
Country	US
Kind code	B2
Filing date	Nov 12, 2012
Priority date	May 11, 2010
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

Abstract

A polynucleotide encoding a biosensor polypeptide comprising a modified circularly-permuted thermostable luciferase and a linker linking the C-terminal portion of the thermostable luciferase to the N-terminal portion of the thermostable luciferase. The modified circularly-permuted thermostable luciferase is modified relative to a parental circularly-permuted thermostable luciferase. The linker contains a sensor region capable of interacting with a target molecule in a cell. The modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to the parental circularly-permuted thermostable luciferase in the presence of the target molecule. Alternatively, the modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to the modified circularly-permuted thermostable luciferase in the absence of the target molecule.

First claim

What is claimed is:

1. A polynucleotide encoding a circularly-permuted thermostable luciferase comprising a peptide linker linking the original N- and C-termini of the non-permuted thermostable luciferase, wherein the peptide linker comprises a granzyme B recognition site, wherein the circularly-permuted thermostable luciferase has increased luminescence following cleavage by granzyme B when compared to an uncleaved circularly-permuted thermostable luciferase, wherein the granzyme B recognition site is selected from the group consisting of GRIEADSE (SEQ ID NO:80), KSVGPDFG (SEQ ID NO:81), and GIETDSG (SEQ ID NO:82), and wherein the non-permuted thermostable luciferase has at least 90% identity to the amino acid sequence of SEQ ID NO:2 or at least 90% identity to the amino acid sequence of SEQ ID NO:4.

2. The polynucleotide of claim 1 , wherein the granzyme B recognition site is flanked by peptide linkers.

3. A vector comprising the polynucleotide of claim 1 .

4. A cell comprising the vector of claim 3 .

5. A kit comprising the polynucleotide of claim 1 .

Assignees

• Promega Corp

Inventors

• Binkowski Brock
• Butler Braeden
• Encell Lance P
• Fan Frank
• Hook Brad
• Otto Paul
• Risma Kimberly
• Vidugiris Gediminas
• Wigdal Susan
• Zimmerman Kristopher

Classifications

• C12N9/0069

  acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13) · CPC title

• C12Q1/66 Primary

  involving luciferase · CPC title

• C12Q1/37

  involving peptidase or proteinase · CPC title