| 摘要 |
A modified luciferase protein which is a sensor for molecules including cAMP, cGMP, calcium, chelators thereof, kinases, or phosphatases is provided. Also provided is a circularly permuted anthozoan luciferase protein and a decapod crustacean luciferase protein, optionally containing one or more heterologous amino acid sequences, at least one of which directly or indirectly interacts with a molecule of interest. Further provided is a modified anthozoan luciferase protein and a decapod crustacean luciferase protein containing an insertion of one or more heterologous amino acid sequences, at least one of which directly or indirectly interacts with a molecule of interest. |
| 主权项 |
1. An isolated polynucleotide comprising a nucleic acid sequence encoding a circularly permuted luciferase, the circularly permuted luciferase comprising a cyclic nucleotide binding site, which circularly permuted luciferase being permuted at a site or in a region which in a corresponding nonpermuted luciferase is tolerant to modification, and wherein luciferase activity of the circularly permuted luciferase is detectable, wherein the cyclic nucleotide binding site comprises at least a portion of a protein selected from the group consisting of exchange protein directly activated by cAMP (Epac), cyclic nucleotide gated ion channels, neuropathy target esterase, PKA regulatory type IIβ subunit, PKA regulatory type Iα subunit, catabolite activating protein, cGMP dependent protein kinase (GK), GAF regulatory region in phosphodiesterase, adenyl cyclases and FnlA, wherein the permutation is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70 to 88, residue 102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to 313, residue 353 to 408, residue 485 to 495, or residue 535 to 546 of a firefly luciferase of SEQ ID NO: 210, a region corresponding to residue 15 to 30, residue 112 to 122, residue 352 to 362, residue 371 to 384, residue 393 to 414, or residue 485 to 495 of a click beetle luciferase of SEQ ID NO: 3, or a region corresponding to residue 2 to 12, residue 26 to 47, residue 64 to 74, residue 85 to 116, residue 147 to 157, residue 223 to 234, or residue 301 to 311 of a Renilla luciferase of SEQ ID NO: 308. |
