The enzyme β-glucuronidase (GUS; E.C.3.2.1.31) hydrolyzes a wide variety of glucuronides. Virtually any aglycone conjugated to D-glucuronic acid through a β-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 β-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, β-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 β-glucuronides, and gusC encoding an outer membrane protein (OMP) that facilitates access of glucuronides to the permease located in the inner membrane.
While β-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, β-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 β-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 calorimetric 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 β-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.
USPTO Backgrounds
USPTO Backgrounds