Microbial B-glucuronidase genes, gene production and uses thereof
AssigneeCambia
InventorsRichard A. Jefferson, Rebecca L. Harcourt, Andrzej Kilian, Katherine Joanna Wilson, Paul Konrad Keese
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Abstract
Genes encoding microbial β-glucuronidase and protein that is secreted and its uses are provided.
Claims83
An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a secreted form of microbial β-glucuronidase.
An isolated secreted form of microbial β-glucuronidase.
An isolated nucleic acid molecule encoding a membrane-bound form of microbial β-glucuronidase.
An isolated nucleic acid molecule encoding a cytoplasmic form of microbial β-glucuronidase.
A vector, comprising a nucleic acid molecule encoding a microbial β-glucuronidase, wherein the β-glucuronidase sequence is in operative linkage with a promoter element.
A method of producing a secreted form of microbial β-glucuronidase, comprising:
- (a) introducing a vector encoding a secreted form of microbial β-glucuronidase in operative linkage with a promoter;
- and
- (b) growing the host cell under conditions wherein the microbial β-glucuronidase is expressed.
A method of introducing a controller element into a host cell, comprising introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial β-glucuronidase and a nucleic acid sequence of the controller element,
- wherein the nucleic acid sequence encoding the β-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1, and which encodes a functional beta-glucuronidase, and wherein the nucleic acid sequence encoding β-glucuronidase is in operative linkage with the controller element.
A method of monitoring expression of a gene of interest or a portion thereof in a host cell, comprising:
- (a) introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial β-glucuronidase and nucleic acid sequence encoding a product of the gene of interest or a portion thereof;
- wherein the nucleic acid sequence encoding the microbial β-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 and which encodes a functional β-glucuronidase, and
- (b) detecting the presence of secreted microbial β-glucuronidase, thereby monitoring expression of the gene of interest.
A method of monitoring activity of a controller element in a host cell, comprising:
- (a) introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial β-glucuronidase and a nucleic acid sequence of the controller element;
- wherein the nucleic acid sequence encoding the β-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 and which encodes a functional β-glucuronidase, and
- wherein the nucleic acid sequence encoding β-glucuronidase is in operative linkage with the controller element;
- and
- (b) detecting the presence of secreted β-glucuronidase, thereby monitoring activity of the controller element.
A method for transforming a host cell with a gene of interest or portion thereof, comprising:
- (a) introducing into the host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a secreted form of microbial β-glucuronidase and nucleic acid sequence encoding a product of the gene of interest or a portion thereof, such that the vector construct integrates into the genome of the host cell;
- wherein the nucleic acid sequence encoding β-glucuronidase (i) encodes a protein comprising the amino acid sequence of FIG. 3 or (ii) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 and which encodes a functional β-glucuronidase;
- and
- (b) detecting the presence of secreted β-glucuronidase, thereby establishing that the host cell is transformed.
A method of positive selection for a transformed cell, comprising:
- (a) introducing into a host cell a vector construct, the vector construct comprising nucleic acid sequence encoding a microbial β-glucuronidase;
- wherein the nucleic acid sequence encoding β-glucuronidase (a) encodes a protein comprising the amino acid sequence of FIG. 3 or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 and which encodes a functional β-glucuronidase;
- and
- (b) exposing the host cell to the sample comprising a glucuronide, wherein the glucuronide is cleaved by the β-glucuronidase, such that the compound is released, wherein the compound is required for cell growth.
A method for identifying an organism that secretes β-glucuronidase, comprising:
- (a) culturing the organism or cells from the organism in a medium containing a substrate for β-glucuronidase, wherein the cleaved substrate is detectable;
- and
- (b) detecting the cleaved substrate in the medium;
- therefrom identifying an organism that secretes β-glucuronidase.
A method for releasing a compound from a glucuronide in a sample, comprising exposing the sample to a microbial β-glucuronidase, wherein the glucuronide is cleaved by the β-glucuronidase, therefrom releasing the compound, provided that the microbial β-glucuronidase is not wild-type E. coli β-glucuronidase.
A method of releasing a compound from a glucuronide exposed to a host cell, comprising:
- (a) introducing into the host cell a vector construct, the vector construct comprising a nucleic acid molecule encoding a microbial β-glucuronidase;
- and
- (b) exposing the host cell to the glucuronide, wherein the glucuronide is cleaved by the beta-glucuronidase, such that the compound is released.
A method of detecting binding between two or more molecules, comprising:
- (a) contacting the two or more molecules in a reaction mixture for a time sufficient to allow binding, wherein one of the molecules is conjugated to a hapten;
- (b) contacting the bound molecules from step (a) with a microbial β-glucuronidase, wherein the β-glucuronidase binds to the hapten;
- and
- (c) detecting the β-glucuronidase.
A method of detecting binding between two or more molecules, comprising:
- (b) contacting the two or more molecules in a reaction mixture for a time sufficient to allow binding, wherein one of the molecules is conjugated to microbial β-glucuronidase;
- (c) detecting the microbial β-glucuronidase;
- therefrom detecting binding between the two molecules.
A kit, comprising a microbial β-glucuronidase, provided that the microbial β-glucuronidase is not E. coli glucuronidase.
Dependencies are inferred from the claim text.