Glucuronide repressors and uses thereof
AssigneeCambia
InventorsRichard A. Jefferson, Katherine J. Wilson
Life of this patent
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Abstract
Clones containing a sequence encoding a glucuronide repressor are described. The nucleotide and amino acid sequences of a repressor (gusR) are presented. A glucuronide repressor is used to control expression of a transgene, detect glucuronides in a sample, and isolate glucuronides from a sample, among other uses.
Claims49
An isolated nucleic acid molecule encoding a glucuronide repressor.
An isolated nucleic acid molecule encoding a domain of a glucuronide repressor that binds a glucuronidase operator.
An isolated nucleic acid molecule encoding a domain from a glucuronide repressor that binds a glucuronide.
An isolated glucuronide repressor that binds to a glucuronide operator and binds to a glucuronide, wherein the binding to the operator is dependent on binding to a glucuronide.
An isolated glucuronide repressor, comprising the amino acid sequence of SEQ ID NO:
- 2 or variant thereof.
An isolated protein, comprising a glucuronide binding domain from a glucuronide repressor.
An isolated protein, comprising a domain from a glucuronide repressor that binds a glucuronidase operator.
A fusion protein comprising a glucuronide binding domain from a glucuronide repressor and a DNA-binding domain that binds to a selected nucleotide sequence.
A transformed host cell containing a nucleic acid molecule that encodes a glucuronide repressor.
A method for determining the presence of a glucuronide in a sample, comprising:
- (a) binding a glucuronide repressor to a nucleic acid molecule comprising a glucuronide operator to form a complex;
- (b) contacting the complex with a sample containing a glucuronide, wherein the glucuronide binds to the repressor protein causing release of the protein from the nucleic acid molecule;
- and
- (c) detecting release of the repressor protein.
A method for controlling gene expression of a transgene introduced into a host cell, comprising:
- (a) transfecting or transforming the host cell with a nucleic acid molecule encoding a glucuronide repressor, a glucuronide operator, and a transgene, wherein the operator is operably linked to the transgene;
- and
- (b) contacting the host cell with a glucuronide that binds to the repressor protein;
- wherein the glucuronide causes the repressor protein to release from the operator, thereby allowing expression of the transgene.
A method for isolating a glucuronide transport protein, comprising:
- (a) transfecting or transforming a cell that does not transport a glucuronide with a nucleic acid molecule encoding a glucuronide repressor and a reporter gene linked to a glucuronide operator;
- (b) further transfecting or transforming the cell in step (a) with an expression library from a vertebrate;
- (c) treating the cell in step (b) with a glucuronide;
- and
- (d) detecting expression of the reporter gene, thereby identifying a cell expressing a transport protein.
A method for isolating a glucuronide repressor protein, comprising:
- (a) contacting the repressor protein with a matrix conjugated with a glucuronide to bind the repressor protein to the glucuronide;
- and
- (b) eluting the repressor protein, thereby isolating the repressor protein.
Dependencies are inferred from the claim text.