US 9822392 B2 - Co-translational Activation Of A Transcription Factor By Proteolytic Cleavage And Methods Of Use - The Lens

Co-translational Activation Of A Transcription Factor By Proteolytic Cleavage And Methods Of Use

Published: Nov 21, 2017
Earliest Priority: Jun 28 2013
Family: 2
Cited Works: 62
Cited by: 0
Cites: 0
Sequences: 22
Additional Info: Cited Works Full text Published Sequence

Abstract

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.

Claims

A method for measuring expression of an autoregulatory molecule, comprising:
expressing a construct in a cell, wherein the construct comprises a purified and isolated polynucleotide, comprising
a polynucleotide sequence encoding an autoregulatory molecule,

a polynucleotide sequence encoding a measurable marker, and

a polynucleotide sequence encoding a cleavable substrate, wherein the polynucleotide sequence encoding the cleavable substrate connects the polynucleotide sequence encoding the autoregulatory molecule and the polynucleotide sequence encoding the measurable marker;

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.
The method of claim 1, wherein the cell is an E. coli cell.
The method of claim 1, wherein the autoregulatory molecule is CI.
The method of claim 1, wherein the measurable marker is selected from the group consisting of fluorescent peptides, colorimetric compounds, chemiluminescent peptides, and combinations thereof.
The method of claim 4, where the fluorescent peptide is selected from the group consisting of yellow fluorescent protein (YFP), blue fluorescent protein (BFP), green fluorescent protein (GFP), red fluorescent protein (RFP) and fluorescing mutants thereof.
A purified and isolated polynucleotide, comprising
a polynucleotide sequence encoding an autoregulatory molecule,

a polynucleotide sequence encoding a measurable marker, and

a polynucleotide sequence encoding a cleavable substrate, wherein the polynucleotide sequence encoding the cleavable substrate connects the polynucleotide sequence encoding the autoregulatory molecule and the polynucleotide sequence encoding the measurable marker, and wherein upon translation of the polynucleotide, when expressed in a cell, the cleavable substrate is cleaved by a protease releasing the measurable marker and allowing the autoregulatory molecule to fold functionally.