Patent ID: 8673558
Filing Date: 2014-03-18
Classification: C07K,C12N,C12Q,G01N

Abstract:
1. A method to detect a molecule of interest in a cell, comprising: a) contacting a cell with a polynucleotide encoding a modified beetle luciferase, wherein the modified beetle luciferase comprises an internal insertion of a heterologous amino acid sequence which interacts with a molecule of interest, which insertion is at a residue or in a region in a beetle luciferase sequence which is tolerant to modification, wherein activity of the modified beetle luciferase is altered after the molecule of interest interacts with the insertion sequence relative to the activity before the interaction, wherein the modified beetle luciferase is a firefly or click beetle luciferase, wherein if the modified beetle luciferase is a firefly luciferase, the insertion is in a region corresponding to residue 116 to 126, residue 228 to 238, residue 262 to 272, residue 289 to 308, residue 356 to 366, or residue 535 to 546 of a firefly luciferase, and wherein if the modified beetle luciferase is a click beetle luciferase, the insertion is in 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; and b) detecting the activity of the modified beetle luciferase encoded by the polynucleotide, thereby detecting the presence of the molecule in the cell.