Title

Microbial B-glucuronidase genes, gene production and uses thereof

Claims

An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a secreted form of microbial β-glucuronidase.
The nucleic acid molecule of claim 1, wherein the microbe is Bacillus.
The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises nucleotides 1662-3467 of FIG. 1 or hybridizes under stringent conditions to the complement of the sequence comprising nucleotides 1662-3467 of FIG. 1, and which encodes a functional β-glucuronidase.
The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes the amino acid sequence of FIG. 3, or a variant thereof, and which encodes a functional β-glucuronidase.
The nucleic acid molecule of claim 4, further comprising a second nucleic acid molecule that encodes the amino acid sequence of FIG. 4A, or a variant thereof, wherein the second nucleic acid molecule is fused to the 5′ end of the molecule of claim 4.
The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes a low-cysteine variant of E. coli β-glucuronidase.
The nucleic acid molecule of claim 6, wherein at least one of the cysteine residues at 28, 133, 197, 253, and 262 are changed.
An isolated secreted form of microbial β-glucuronidase.
The β-glucuronidase of claim 8, wherein the microbe is Bacillus.
The β-glucuronidase of claim 8, wherein the β-glucuronidase comprises a low-cysteine variant of E. coli β-glucuronidase.
The β-glucuronidase of claim 10, wherein the β-glucuronidase has at least one alteration of the cysteine residue at 28, 133, 197, 253, and 262.
The β-glucuronidase of claim 9, wherein the β-glucuronidase is encoded by a nucleic acid molecule comprising nucleotides 1662-3467 of FIG. 1 or by a nucleic acid molecule that hybridizes under stringent conditions to the complement of nucleotides 1662-3467 of FIG. 1 and which encodes a functional β-glucuronidase.
The β-glucuronidase of claim 9, comprising the amino acid sequence of FIG. 3, or a variant thereof, and which encodes a functional β-glucuronidase.
An isolated nucleic acid molecule encoding a membrane-bound form of microbial β-glucuronidase.
The nucleic acid molecule of claim 14, wherein the sequence encoding β-glucuronidase is fused to a sequence encoding a membrane-spanning peptide.
An isolated nucleic acid molecule encoding a cytoplasmic form of microbial β-glucuronidase.
The nucleic acid molecule of claim 16, wherein the sequence encoding β-glucuronidase is fused to a sequence encoding a cytoplasm-directing peptide.
The nucleic acid molecule of claim 17, wherein the cytoplasm-directing peptide is Lys-Asp-Glu-Leu.
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.
The vector of claim 19, wherein the promoter element is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter.
The vector of claim 20, wherein the promoter element is selected from a group consisting of a promoter functional in a plant cell, a promoter functional in a bacterium, a promoter functional in an animal cell and a promoter functional in a fungal cell.
The vector of claim 19, wherein the vector is functional in a bacterium.
The vector of claim 19, wherein the vector is a binary Agrobacterium tumefaciens plasmid vector.
The vector of claim 19, further comprising a nucleic acid sequence encoding a product of a gene of interest or portion thereof.
The vector of claim 24, wherein the product is a protein.
A host cell containing the vector according to claim 19.
The host cell of claim 26, wherein the host cell is selected from the group consisting of a plant cell, an insect cell, a fungal cell, an animal cell and a bacterial cell.
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.
The method according to claim 28, further comprising isolating the β-glucuronidase from cell supernatant or periplasm.
The method according to claim 28, wherein the host cell is a bacterial cell.
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.
The method according to claim 31, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell.
The method according to claim 31, wherein the vector construct is a binary Agrobacterium vector.
The method according to claim 31, wherein the controller element is selected from the group consisting of a promoter, an enhancer, an operator, a ribosome binding site, a signal peptide sequence, a chloroplast targeting sequence, a mitochondrial localization sequence, a nucleus targeting sequence and an intron.
The method according to claim 34, wherein the controller element is functional in a plant cell.
The method according to claim 34, wherein the controller element is a promoter selected from the group consisting of a developmental type-specific promoter, a tissue type-specific promoter, a cell type-specific promoter and an inducible promoter.
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.
The method according to claim 37, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell.
The method according to claim 37, wherein the product is a protein.
The method according to claim 37, wherein the vector construct further comprises a promoter.
The method according to claim 37, wherein the nucleic acid sequence encoding the product and the nucleic acid sequence encoding β-glucuronidase are in operative linkage with the same promoter.
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.
The method according to claim 42, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell.
The method according to claim 42, wherein the vector construct is a binary Agrobacterium vector.
The method according to claim 42, wherein the controller element is selected from the group consisting of a promoter, an enhancer, an operator, a ribosome binding site, a signal peptide sequence, a chloroplast targeting sequence, a mitochondrial localization sequence, a nucleus targeting sequence and an intron.
The method according to claim 42, wherein the controller element is a promoter functional in a plant cell.
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.
The method according to claim 47, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell.
The method according to claim 47, wherein the vector construct is a binary Agrobacterium vector.
The method according to claim 47, wherein the product is a protein.
The method according to claim 47, wherein the vector construct further comprises a promoter.
The method according to claim 47, wherein the gene of interest and β-glucuronidase are under control of the same promoter.
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.
The method of claim 53, further comprising introducing into the host cell a vector construct comprising a nucleic acid sequence encoding a microbial glucuronide permease.
The method according to claim 53, wherein the host cell is selected from the group consisting of a plant cell, an animal cell, an insect cell, a fungal cell and a bacterial cell.
A method of producing a transgenic plant that expresses a secreted form of microbial β-glucuronidase, comprising:
(a) introducing a vector according to claim 19 into an embryogenic plant cell; and

(b) producing a plant from the embryogenic plant cell, wherein the plant expresses the β-glucuronidase.
The method of claim 56, wherein the step of introducing is by Agrobacterium co-cultivation or bombardment.
A transgenic plant cell comprising the vector according to claim 19.
A transgenic plant comprising the plant cell of claim 58.
A transgenic insect cell comprising the vector according to claim 19.
A transgenic insect comprising the insect cell of claim 60.
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.
The method of claim 62, wherein the organism is a bacterium isolated from soil, skin, or fecal matter.
The method of claim 62, wherein the organism is Bacillus.
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.
The method of claim 65, further comprising detecting the compound.
The method of claim 65, wherein the β-glucuronidase comprises the amino acid sequence presented in FIG. 3.
The method of claim 65, wherein the sample is a biological fluid selected from the group consisting of blood, saliva, urine, apocrine secretion, synovial fluid and amniotic fluid.
The method of claim 65, wherein the compound is a toxin, a hormone, or a drug.
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.
The method of claim 70, wherein the nucleic acid molecule encoding beta-glucuronidase (a) encodes a protein comprising the amino acid sequence as depicted in FIG. 3 or (b) hybridizes under stringent conditions to the complement of nucleotides 1662-3467 as depicted in FIG. 1 and which encodes a functional beta-glucuronidase.
The method of claim 70, further comprising introducing into the host cell a vector construct comprising a nucleic acid molecule encoding a glucuronide permease.
The method according to claim 70, wherein the compound is an auxin.
The method according to claim 73, wherein the auxin is indole-3-ethanol.
The method according to claim 70, wherein the compound is a hormone or a toxin.
The method according to claim 70, wherein the compound is required for cell growth.
The method according to claim 70, wherein the host cell is a plant cell, an animal cell, a fungal cell, or a bacterial cell.
The method according to claim 70, wherein the compound is a herbicide.
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.
The method of claim 79, wherein the β-glucuronidase is fused to a protein that binds the hapten.
The method of claim 80, wherein the protein is streptavidin and the hapten is biotin.
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.

Abstract

Genes encoding microbial β-glucuronidase and protein that is secreted and its uses are provided.

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/058,263, filed Sep. 9, 1997, which application is incorporated by reference in its entirety.

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