CO-TRANSLATIONAL ACTIVATION OF A TRANSCRIPTION FACTOR BY PORTEOLYTIC CLEAVAGE AND METHODS OF USE

摘要

A method for measuring expression of autoregulatory molecules within living cells is provided. An autoregulatory molecule and marker construct is expressed in vivo, where the marker is cleaved from the construct during translation. The method comprises the expression of a construct having an autoregulatory molecule bound to a measurable expression marker by a cleavable linker. The cleavable linker is the substrate of a protease, which acts on its substrate in vivo during translation. Cleavage during translation, allows the autoregulatory molecule to fold normally as it would in its native form. The measurable marker is released and available for detection upon cleavage by the protease. As a result, the concentration of the measurable marker is directly related to the level of expression of the autoregulatory molecule.

主权项

1. A method for measuring expression of an autoregulatory molecule, comprising:
expressing a construct in a cell, wherein the construct comprises the autoregulatory molecule, a measurable marker, and a cleavable substrate in a single molecule; expressing a protease capable of cleaving the cleavable substrate in the cell, wherein the protease cleaves the cleavable substrate during translation allowing the autoregulatory molecule to fold into a functional molecule; and evaluating the cell for the presence of the measurable substrate.