发明名称 |
Mutant protease biosensors with enhanced detection characteristics |
摘要 |
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. |
申请公布号 |
US9290794(B2) |
申请公布日期 |
2016.03.22 |
申请号 |
US201213674655 |
申请日期 |
2012.11.12 |
申请人 |
PROMEGA CORPORATION |
发明人 |
Binkowski Brock;Butler Braeden;Encell Lance P.;Fan Frank;Hook Brad;Otto Paul;Risma Kimberly;Vidugiris Gediminas;Wigdal Susan;Zimmerman Kristopher |
分类号 |
C12Q1/66;C12Q1/37;C12N9/02 |
主分类号 |
C12Q1/66 |
代理机构 |
Michael Best & Friedrich LLP |
代理人 |
Michael Best & Friedrich LLP |
主权项 |
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. |
地址 |
Madison WI US |