Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-17
2003-11-04
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S207000, C435S252300, C435S320100, C435S471000, C536S023100, C536S024100
Reexamination Certificate
active
06641996
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to microbial &bgr;-glucuronidases, and more specifically to secreted forms of &bgr;-glucuronidase, and uses of these &bgr;-glucuronidases.
BACKGROUND OF THE INVENTION
The enzyme &bgr;-glucuronidase (GUS; E.C.3.2.1.31) hydrolyzes a wide variety of glucuronides. Virtually any aglycone conjugated to D-glucuronic acid through a &bgr;-O-glycosidic linkage is a substrate for GUS. In vertebrates, glucuronides containing endogenous as well as xenobiotic compounds are generated through a major detoxification pathway and excreted in urine and bile.
Escherichia coli,
the major organism resident in the large intestine of vertebrates, utilizes the glucuronides generated in the liver and other organs as an efficient carbon source. Glucuronide substrates are taken up by
E. coli
via a specific transporter, the glucuronide permease (U.S. Pat. Nos. 5,288,463 and 5,432,081), and cleaved by &bgr;-glucuronidase, releasing glucuronic acid residues that are used as a carbon source. In general, the aglycone component of the glucuronide substrate is not used by
E. coli
and passes back across the bacterial membrane into the gut to be reabsorbed into the bloodstream and undergo glucuronidation in the liver, beginning the cycle again. In
E. coli
, &bgr;-glucuronidase is encoded by the gusA gene (Novel and Novel,
Mol. Gen. Genet.
120:319-335, 1973), which is one member of an operon comprising two other protein-encoding genes, gusB encoding a permease (PER) specific for &bgr;-glucuronides, and gusC encoding an outer membrane protein (OMP) that facilitates access of glucuronides to the permease located in the inner membrane.
While &bgr;-glucuronidase activity is expressed in almost all tissues of vertebrates and their resident intestinal flora, GUS activity is absent in most other organisms. Notably, plants, most bacteria, fungi, and insects are reported to largely, if not completely, lack GUS activity. Thus, GUS is ideal as a reporter molecule in these organisms and has become one of the most widely used reporter systems for these organisms.
In addition, because both endogenous and xenobiotic compounds are generally excreted from vertebrates as glucuronides, &bgr;-glucuronidase is widely used in medical diagnostics, such as drug testing. In therapeutics, GUS has been used as an integral component of prodrug therapy. For example, a conjugate of GUS and a targeting molecules, such as an antibody specific for a tumor cell type, is delivered along with a nontoxic prodrug, provided as a glucuronide. The antibody targets the cell and GUS cleaves the prodrug, releasing an active drug at the target site.
Because the
E. coli
GUS enzyme is much more active and stable than the mammalian enzyme against most biosynthetically derived &bgr;-glucuronides (Tomasic and Keglevic,
Biochem J
133:789, 1973; Levvy and Conchie, 1966), the
E. coli
GUS is preferred in both reporter and medical diagnostic systems.
Production of GUS for use in in vitro assays, such as medical diagnostics, however, is costly and requires extensive manipulation as GUS must be recovered from cell lysates. A secreted form of GUS would reduce manufacturing expenses, however, attempts to cause secretion have been largely unsuccessful. In addition, for use in transgenic organisms, the current GUS system has somewhat limited utility because enzymatic activity is detected intracellularly by deposition of toxic colorimetric products during the staining or detection of GUS. Moreover, in cells that do not express a glucuronide permease, the cells must be permeabilized or sectioned to allow introduction of the substrate. Thus, this conventional staining procedure generally results in the destruction of the stained cells. In light of these limitations, a secreted GUS would facilitate development of non-destructive marker systems, especially useful for agricultural field work.
Furthermore, the
E. coli
enzyme, although more robust than vertebrate GUS, has characteristics that limit its usefulness. For example, it is heat-labile and inhibited by detergents and end product (glucuronic acid). For many applications, a more resilient enzyme would be beneficent.
The present invention provides gene and protein sequences of microbial &bgr;-glucuronidases, variants thereof, and use of the proteins as a transformation marker, effector molecule, and component of medical diagnostic and therapeutic systems, while providing other related advantages.
SUMMARY OF INVENTION
In one aspect, an isolated nucleic acid molecule is provided comprising a nucleic acid sequence encoding a microbial of &bgr;-glucuronidase, provided that the &bgr;-glucuronidase is not from
E. coli.
Nucleic acid sequences are provided for &bgr;-glucuronidases from Thermotoga, Bacillus, Staphylococcus, Salmonella, Enterobacter, and Pseudomonas. In certain embodiments, the nucleic acid molecule encoding &bgr;-glucuronidase is derived from a eubacteria, such as purple bacteria, gram(+) bacteria, cyanobacteria, spirochaetes, green sulphur bacteria, bacteroides and flavobacteria, planctomyces, chlamydiae, radioresistant micrococci, and thermotogales.
In another aspect, microbial &bgr;-glucuronidases are provided that have enhanced characteristics. In one aspect, thermostable &bgr;-glucuronidases and nucleic acids encoding them are provided. In general, a thermostable &bgr;-glucuronidase has a half-life of at least 10 min at 65° C. In preferred embodiments, the thermostable &bgr;-glucuronidase is from Thermotoga or Bacillus groups. In other embodiments, the &bgr;-glucuronidase converts at least 50 nmoles of p-nitrophenyl-glucuronide to p-nitrophenyl per minute, per microgram of protein. In even further embodiments, the &bgr;-glucuronidase retains at least 80% of its activity in 10 mM glucuronic acid.
In another aspect, fusion proteins of microbial &bgr;-glucuronidase or an enzymatically active portion thereof are provided. In certain embodiments, the fusion partner is an antibody or fragment thereof that binds antigen.
In other aspects, expression vectors comprising a gene encoding a microbial &bgr;-glucuronidase or a portion thereof that has enzymatic activity in operative linkage with a heterologous promoter are provided. In such a vector, the microbial &bgr;-glucuronidase is not
E. coli
&bgr;-glucuronidase. In the expression vectors, the heterologous promoter 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 promoter should be functional in the host cell for the expression vector. Examples of cell types include a plant cell, a bacterial cell, an animal cell and a fungal cell. In certain embodiments, the expression vector also comprises a nucleic acid sequence encoding a product of a gene of interest or portion thereof. The gene of interest may be under control of the same or a different promoter.
Isolated forms of recombinant microbial &bgr;-glucuronidase are also provided in this invention, provided that the microbial &bgr;-glucuronidase is not
E. coli
&bgr;-glucuronidase. The recombinant &bgr;-glucuronidases may be from eubacteria, archaea, or eucarya. When eubacteria &bgr;-glucuronidases are clones, the eubacteria is selected from purple bacteria, gram(+) bacteria, cyanobacteria, spirochaetes, green sulphur bacteria, bacteroides and flavobacteria, planctomyces, chlamydiae, radioresistant micrococci, and thermotogales and the like.
The present invention also provides methods for 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 a nucleic acid molecule according to claim 1 and a nucleic acid molecule encoding a product of the gene of interest or a portion thereof; (b) detecting the presence of the microbial &bgr;-glucuronidase, thereby monitoring expression of the gene of interest; methods for transforming a host cell with a gene of interest or portion thereof, compr
Jefferson Richard A.
Mayer Jorge E.
Cambia
Sandals William
Yucel Remy
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