HK1189157B - Crystallized oxalate decarboxylase and methods of use - Google Patents

Description

Crystallized oxalate decarboxylase and methods of use

This application is a divisional application of Chinese patent application No. 200780035792.4 (PCT/US2007/075091), filed on August 2, 2007, entitled "Crystallized oxalate decarboxylase and methods of use"

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application Serial No. 60/834,933, filed August 2, 2006, and U.S. Application Serial No. 60/854,540, filed October 26, 2006, the contents of which are incorporated herein by reference in their entireties.

Background Art

Oxalic acid is a dicarboxylic acid with the formula _HO₂C — _CO₂H . Oxalic acid exists primarily in organisms as oxalates, which are salt forms of oxalic acid. Oxalates are found in foods such as spinach, rhubarb, strawberries, cranberries, nuts, cocoa, chocolate, peanut butter, sorghum, and tea. Oxalate is also a metabolic end product in humans and other mammals. It is excreted in the urine by the kidneys. When combined with calcium, oxalic acid forms the insoluble product calcium oxalate, the most common compound found in kidney stones.

Because mammals do not synthesize enzymes that degrade oxalate, oxalate levels in individuals are generally maintained normal by excretion and low absorption of dietary oxalate. High concentrations of oxalate are associated with a variety of pathologies, such as primary hyperoxaluria, enteric hyperoxaluria, and idiopathic hyperoxaluria. Leumann et al., Nephrol.Dial.Transplant. 14 : 2556-2558 (1999) and Earnest, Adv.Internal Medicine 24 : 407-427 (1979). The causes of increased oxalate can be excessive oxalate intake from food, excessive absorption of oxalate from the intestine, and abnormalities in oxalate production. Excessive absorption of oxalate in the colon and small intestine is associated with enteropathy, including excessive absorption caused by bile acid and fat malabsorption diseases; ileal resection; and, for example, steatorrhea caused by celiac disease, exocrine pancreatic insufficiency, enteropathy, and liver disease.

Hyperoxaluria, or increased urinary oxalate excretion, is associated with a number of health problems involving the deposition of calcium oxalate in kidney tissue (nephrocalcinosis) or the urinary tract (e.g., kidney stones, urolithiasis, and nephrolithiasis). Calcium oxalate can also deposit in, for example, the eyes, blood vessels, joints, bones, muscles, heart, and other vital organs, causing damage to them. See, for example, Leumann et al., J. Am. Soc. Nephrol. 12 :1986-1993 (2001) and Monico et al., Kidney International 62 :392-400 (2002). The effects of increased oxalate levels can occur in a variety of tissues. For example, deposition in small blood vessels can cause painful skin ulcers that are difficult to heal, deposition in the bone marrow can cause anemia, deposition in bone tissue can cause fractures or affect children's growth, and calcium oxalate deposition in the heart can cause abnormal heart rate or poor heart function.

Existing methods for treating elevated oxalate levels are not always effective, and many patients with primary hyperoxaluria may require intensive dialysis and organ transplantation. Existing therapies for different hyperoxalurias include high-dose vitamin B6, orthophosphate, magnesium, iron, aluminum, potassium citrate, cholestyramine, and glycosaminoglycan treatments, as well as regimens for adjusting diet and fluid intake, dialysis, and surgery (e.g., kidney and liver transplantation). These therapies (e.g., low oxalate or low-fat diets, vitamin B6, sufficient calcium, and increased fluids) are only partially effective and may have undesirable adverse side effects, such as the gastrointestinal effects of orthophosphate, magnesium, or cholestyramine supplementation, and the risks of dialysis and surgery. Therefore, there is a need for methods for safely removing oxalate from the body. In addition, methods for degrading oxalate to reduce oxalate levels in biological samples outperform therapies such as blocking oxalate absorption or accelerating oxalate clearance alone.

Summary of the Invention

The present invention relates to oxalate decarboxylase ("OXDC") crystals and cross-linked forms thereof ("CLEC") and their use for treating oxalate-related conditions such as hyperoxaluria. In one embodiment, crystalline oxalate decarboxylase can be administered to a mammal, for example, orally or directly into the stomach, to reduce oxalate levels and/or reduce the damage caused by calcium oxalate deposition in the mammal. Additionally, methods for producing protein crystals from cell extracts are disclosed. Compositions, for example, pharmaceutical compositions, comprising oxalate decarboxylase ("OXDC") crystals and cross-linked forms thereof ("CLEC") are also disclosed.

In one aspect, the invention provides cross-linked oxalate decarboxylase crystals. The cross-linking agent can be multifunctional, and in certain embodiments, the agent is a bifunctional agent, such as glutaraldehyde. In certain embodiments, the oxalate decarboxylase crystals are cross-linked with glutaraldehyde that does not substantially change the concentration of enzyme activity, such as a concentration of at least about 0.02% (w/v). In embodiments, the cross-linking level of the oxalate decarboxylase crystals is equal to the level produced by treatment with 0.02% (w/v) glutaraldehyde. The cross-linking level can be measured by methods known in the art or disclosed herein, for example, by measuring the level of protein extraction, such as disclosed in Examples 10-11.

The present invention also provides oxalate decarboxylase crystals, for example, oxalate decarboxylase crystals having an activity greater than, for example, at least about 100%, 200%, 300%, 400%, or 500% greater than that of soluble oxalate decarboxylase.

The present invention also provides stabilized, e.g., cross-linked, oxalate decarboxylase crystals, wherein the stabilized crystals retain activity and/or stability under acidic conditions that is at least 2 or 3 times greater than the activity and/or stability of a soluble oxalate decarboxylase under similar acidic conditions (e.g., an acidic pH of about 2 to 3). In embodiments, the stabilized oxalate decarboxylase crystals are at least 200%, 300%, or 400% more active and/or stable than a soluble oxalate decarboxylase under acidic conditions.

The present invention also provides stabilized, for example, cross-linked oxalate decarboxylase crystals, wherein the stabilized crystals retain activity and/or stability in the presence of a protease at least 2 or 3 times higher than the activity and/or stability retained by a soluble oxalate decarboxylase under similar conditions. In embodiments, the stabilized oxalate decarboxylase crystals are at least 200%, 300%, or 400% more active and/or stable than a soluble oxalate decarboxylase in the presence of a protease. The protease may be selected from one or more of the following: for example, pepsin, chymotrypsin, or pancreatin. In embodiments, the activity of the stabilized or soluble oxalate decarboxylase is measured after the stabilized crystals or soluble oxalate decarboxylase are exposed to acidic conditions and/or protease for a predetermined length of time, for example, at least 1, 2, 3, 4, or 5 hours.

In a related aspect, the present invention features cross-linked oxalate decarboxylase crystals that are substantially active and stable under various pH conditions (e.g., from about pH 2.5 or 3 to 7.5 or 8.5) and/or in the presence of a protease, such as one or more of pepsin, chymotrypsin, or pancreatin. As described herein, in embodiments, the cross-linked crystals retain activity that is at least 2 or 3 times greater than the activity retained by soluble oxalate decarboxylase under acidic conditions (e.g., an acidic pH of about 2 to 3) and in the presence of a protease. In other embodiments, the stabilized oxalate decarboxylase crystals are at least 200%, 300%, or 400% more stable than soluble oxalate decarboxylase under acidic conditions (e.g., an acidic pH of about 2 to 3) and in the presence of a protease, as described herein.

Compositions, eg, pharmaceutical compositions, comprising crystals and/or cross-linked oxalate decarboxylase crystals described herein are also within the scope of the invention.

In some embodiments, the crystals include an oxalate decarboxylase having a sequence that is identical or substantially identical to an oxalate decarboxylase found in a natural source, such as plants, bacteria, and fungi, particularly Bacillus subtilis, Flammulina velutipes or Flammulina velutipes, Aspergillus niger, Pseudomonas sp., Cyanobacteria, Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum, T. versicolor, Postia placenta, Myroculia verrucosum, Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovoraxparadoxus, Xanthomonas autotrophicus, Aspergillus, Penicillium, and Mucor. In other embodiments, the oxalate decarboxylase is produced recombinantly.

In one aspect, the present invention provides a method for reducing the oxalate concentration in a subject, wherein a composition disclosed herein, e.g., a pharmaceutical composition, comprising oxalate decarboxylase crystals, e.g., cross-linked oxalate decarboxylase crystals, is administered. In one embodiment, the oxalate decarboxylase crystals are stabilized with a cross-linking agent such as glutaraldehyde. Administration of the composition can reduce the oxalate concentration by at least 10%, at least 20%, at least 30%, or at least 40% or more. In some embodiments, the composition is administered orally or via an in vitro device. In one embodiment, the in vitro device is a catheter, e.g., a catheter coated with oxalate decarboxylase crystals. In other embodiments, the composition is administered as a suspension, dry powder, capsule, or tablet. In one embodiment, the method for reducing the oxalate concentration in a mammal comprises the step of determining the oxalate concentration in a biological sample (e.g., a urine, blood, plasma, or serum sample) of the mammal.

In another aspect, the present invention provides a method for treating, preventing, and/or slowing the progression of a condition associated with elevated oxalate concentrations in a mammal, wherein oxalate decarboxylase crystals and/or stabilized, e.g., cross-linked, oxalate decarboxylase crystals are administered to the mammal. In one embodiment, the condition associated with elevated oxalate concentrations is nephropathy, arthritis, eye disease, liver disease, gastrointestinal disease, or pancreatic disease. In certain embodiments, the condition is primary hyperoxaluria, enterogenic hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol poisoning, cystic fibrosis, inflammatory bowel disease, urolithiasis, kidney stones, chronic kidney disease, hemodialysis, and gastrointestinal bypass.

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising oxalate decarboxylase crystals, e.g., cross-linked oxalate decarboxylase crystals (e.g., crystals and/or cross-linked crystals disclosed herein).

In another aspect, the present invention provides a method of treating a mammal by administering an effective amount of a pharmaceutical composition comprising oxalate decarboxylase crystals, e.g., cross-linked oxalate decarboxylase crystals (e.g., crystals and/or cross-linked crystals disclosed herein).

In another aspect, the present invention provides a method for producing protein crystals, such as enzyme crystals (e.g., oxalate decarboxylase crystals), comprising: providing a preparation of cell extracts or particles/precipitates/solutions containing the protein, and crystallizing the protein from the preparation. In embodiments, the method comprises one or more of the following steps: culturing a prokaryotic host cell culture expressing the protein; obtaining a preparation of particles or extracts containing the target protein; solubilizing the particle preparation; allowing protein crystals to form, and/or further stabilizing the crystals by cross-linking. Typically, the protein is recombinantly expressed. In embodiments, the particle preparation comprises inclusion bodies.

In embodiments, the solubilization step comprises adding one or more of the following to the particle preparation: a solution comprising a mild denaturant concentration (e.g., urea or guanidine hydrochloride at a concentration of, for example, about 1 M to about 3 M); a solution comprising a high salt concentration (e.g., the salt is selected from one or more of sodium chloride, potassium chloride, calcium chloride) or other salts at a concentration of, for example, about 0.3 to about 0.8 M; a solution comprising a mild denaturant concentration under alkaline conditions (e.g., at a pH of about 9 to about 12); or a solution comprising a high denaturant concentration (e.g., about 4 M to about 8 M urea or guanidine hydrochloride).

In embodiments, the purification step comprises removing debris from the granules, for example, by isolating the solubilized granule preparation (e.g., by one or more spinning or centrifugation steps) and/or collecting the supernatant. The purification step may optionally further comprise subjecting the solubilized granule preparation to ion exchange chromatography and/or filtering the solubilized preparation.

In embodiments, the crystallization step comprises concentrating the purified protein to form a crystallized protein. In embodiments, the crystallized protein is obtained from the supernatant collected after the separation step, for example, after one or more rotation or centrifugation steps. The crystallization step may further comprise contacting the crystallized protein with a cross-linking agent, for example, a cross-linking agent disclosed herein (e.g., glutaraldehyde). The concentration of the cross-linking agent used may range from 0.01% to 20% w/v; typically 0.02% to 10% w/v; more typically 0.02%, 0.5% or 1% w/v.

In embodiments, the yield of the protein in the granule preparation is at least about 50%, 60%, 70%, or 80% of the particular protein found in the cell preparation from which the granule preparation is derived. In other embodiments, the yield of the solubilized protein is at least about 90%, 95%, or more of the protein found in the granule preparation. In other embodiments, the yield of the crystallized protein is at least about 50%, 60%, 70%, or 80% of the protein found in the granule preparation.

The present invention also provides protein crystals, eg, enzyme crystals (eg, oxalate decarboxylase crystals), produced by the methods disclosed herein.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the pH activity profiles of soluble oxalate decarboxylase ("Soluble"), oxalate decarboxylase crystals ("Crystals"), and cross-linked oxalate decarboxylase crystals ("CLEC").

Figure 2 is a bar graph depicting urine oxalate levels in Sprague Dawley rats after administration of OXDC-CLEC. Each bar represents the mean ± SE (standard error). Asterisks indicate significant differences (p < 0.05) between the control group and the three treatment groups at the indicated time points calculated using a two-tailed Student's t-test.

Figure 3 is a bar graph depicting the effect of OXDC-CLEC on the reduction of urinary oxalate levels in ethylene glycol-challenged AGT1 knockout (KO) mice. Each bar represents the mean ± SE. Asterisks indicate significant differences (p < 0.05) between the control group and the three treatment groups at the indicated time points, calculated using a two-tailed Student's t-test.

Figure 4 is a bar graph depicting the effect of OXDC-CLEC on creatinine clearance in ethylene glycol-challenged AGT1 KO mice. Each bar represents the mean ± SE. Asterisks indicate significant differences between the control group and the 80 mg treatment group (p < 0.05) calculated using a two-tailed Student's t-test.

Figures 5A-5C are images showing the prevention of calcium oxalate deposits in the renal parenchyma following treatment with OXDC-CLEC. Figure 5A is a section of renal parenchyma from an EG-challenged mouse treated with 80 mg of OXDC-CLEC. Figures 5B and 5C are sections of renal parenchyma from a control group. The section in Figure 5B demonstrates moderate nephrocalcinosis, and the section in Figure 5C demonstrates severe nephrocalcinosis. Dark spots are calcium oxalate deposits (examples indicated by white arrows), and light spots are areas of interstitial fibrosis (examples indicated by gray arrows).

FIG6 is a Kaplan-Meier survival graph comparing the survival time of EG-challenged mice treated with three different doses of OXDC-CLEC or vehicle control.

7 is a graph depicting the stability of soluble oxalate decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL"), and cross-linked oxalate decarboxylase crystals ("CLEC") at low pH at the indicated time intervals.

8 is a graph depicting the stability of soluble oxalate decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL"), and cross-linked oxalate decarboxylase crystals ("CLEC") in the presence of pepsin at pH 3.0 at the indicated time intervals.

Figure 9 is a graph depicting the stability of soluble oxalate decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL"), and cross-linked oxalate decarboxylase crystals ("CLEC") in the presence of chymotrypsin at pH 7.5 at the indicated time intervals.

Figure 10 is a graph depicting the stability of soluble oxalate decarboxylase ("Sol"), oxalate decarboxylase crystals ("XTAL"), and cross-linked oxalate decarboxylase crystals ("CLEC") in the presence of simulated intestinal fluid containing pancreatin at pH 6.8 at the indicated time intervals.

Detailed Description of the Invention

The present invention is based, in part, on the discovery that administration of oxalate decarboxylase (OXDC) crystals can alleviate the symptoms of hyperoxaluria in mammals. Methods of administering OXDC crystals to treat various oxalate-related conditions are described herein. Additionally, OXDC crystals and cross-linked crystals (CLECs), compositions containing and using the same, are provided. Furthermore, methods for producing large quantities of protein crystals from cell extracts of prokaryotic host cells are disclosed.

Definitions. To facilitate understanding of the present invention, certain terms are first defined. Additional definitions are set forth in the detailed description.

As used herein, a "biological sample" is a biological material collected from cells, tissues, organs, or organisms, e.g., for the detection of an analyte. Exemplary biological samples include fluids, cell, or tissue samples. Biological fluids include, for example, serum, blood, plasma, saliva, urine, or sweat. Cell or tissue samples include biopsies, tissues, cell suspensions, or other specimens and samples, such as clinical samples.

A "crystal" is a form of solid matter that contains atoms arranged in a three-dimensional, periodically repeating pattern (see, e.g., Barret, Structure of Metals, ^2nd ed., McGraw-Hill, New York (1952)). The crystalline form of a polypeptide, for example, is distinct from the second form, the amorphous solid state. Crystals exhibit unique characteristics, including shape, lattice structure, solvent percentage, and optical properties, e.g., refractive index.

An "extracorporeal device" is a structure outside the body that is used to contact bodily fluids with OXDC crystals in the treatment of an individual. Preferably, the extracorporeal device is a device for dialysis, including renal dialysis, a device for continuous arteriovenous hemofiltration, an extracorporeal membrane oxygenator, or other device for filtering waste products from the bloodstream. Similarly, the term includes components of a device that filters waste products, including, for example, catheters, porous materials, or membranes. More specifically, the extracorporeal device can be a dialysis device. Alternatively, it can be a membrane of a dialysis device.

A "functional fragment" of OXDC is a portion of an OXDC polypeptide that retains one or more biological activities of OXDC, such as the ability to catalyze the decarboxylation of oxalate. As used herein, a functional fragment may comprise a terminal truncation from one or both termini, unless otherwise indicated. For example, a functional fragment may have 1, 2, 4, 5, 6, 8, 10, 12, 15, or 20 or more residues omitted from the amino and/or carboxyl termini of an OXDC polypeptide. Preferably, the truncation does not exceed 20 amino acids from one or both termini. A functional fragment may optionally be linked to one or more heterologous sequences.

The term "individual" or "subject" refers to any mammal, including, but not limited to, any animal so classified, including humans, non-human primates, primates, baboons, gorillas, monkeys, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

The term "isolated" refers to a molecule that is substantially free from its natural environment. For example, an isolated protein is substantially free from cellular material or other proteins from the cell or tissue source from which it was derived. The term refers to a preparation in which the isolated protein is sufficiently pure to be administered as a therapeutic composition.

Or at least 70% to 80% (w/w) pure, more preferably at least 80% to 90% (w/w) pure, more preferably 90 to 95% pure; most preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) pure.

As used herein, the term "about" refers to up to ±10% of the value defined by the term. For example, about 50 mM refers to 50 mM ± 5 mM; about 4% refers to 4% ± 0.4%.

As used herein, "oxalate-related conditions" refer to diseases or conditions associated with pathological levels of oxalic acid or oxalate, including, but not limited to, hyperoxaluria, primary hyperoxaluria, enteric hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol (oxalate) poisoning, idiopathic urolithiasis, renal failure (including progressive, chronic, or end-stage renal failure), steatorrhea, malabsorption, ileopathy, vulvodynia, cardiac conductance disorders, inflammatory bowel disease, cystic fibrosis, pancreatic exocrine insufficiency, Crohn's disease, ulcerative colitis, nephrocalcinosis, urolithiasis, and kidney stones. Such conditions and disorders may optionally be acute or chronic. Oxalate-related conditions associated with the kidneys, bones, liver, gastrointestinal tract, and pancreas are known in the art. Furthermore, it is well known that calcium oxalate can be deposited in a variety of tissues, including, but not limited to, the eyes, blood vessels, joints, bones, muscles, heart, and other vital organs, leading to many oxalate-related conditions.

At the pH of urine and intestinal fluid ( _pKa1 = 1.23, _pKa2 = 4.19), "oxalic acid" exists primarily in its salt form, oxalate (as the salt of the corresponding conjugate base). Earnest, Adv. Internal Medicine 24 : 407427 (1979). The terms "oxalic acid" and "oxalate" are used interchangeably in this disclosure. Oxalates containing lithium, sodium, potassium, and iron (II) are soluble, but calcium oxalate is generally very poorly soluble in water (e.g., only dissolving to about 0.58 mg/100 ml at 18°C. Earnest, Adv. Internal Medicine 24 : 407427 (1979)). Oxalic acid from food is also referred to as dietary oxalate. Oxalate produced by metabolic processes is referred to as endogenous oxalate. Circulating oxalate is oxalate present in circulating body fluids, such as blood.

The term "therapeutically effective dose" or "therapeutically effective amount" refers to an amount of a compound that results in the prevention, delay of symptom onset, or amelioration of symptoms of an oxalate-related condition (including hyperoxaluria, such as primary hyperoxaluria or enteric hyperoxaluria). A therapeutically effective amount is sufficient, for example, to treat, prevent, lessen the severity of, delay the onset of, and/or reduce the risk of onset of one or more symptoms of a condition associated with elevated oxalate concentrations. An effective amount can be determined by methods well known in the art and described in subsequent sections of this specification.

The terms "treatment," "therapeutic method," and their synonyms refer to treatment and/or prophylactic/preventative measures for an existing condition. Subjects in need of treatment can include individuals who already have a particular medical condition, as well as subjects who are at risk for, have, or are likely to eventually develop the condition. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of the disease, the presence or progression of the condition, or the likely receptivity of a subject with the condition to treatment. Treatment can include slowing or reversing the progression of the condition.

Oxalate decarboxylase. As used herein, oxalate decarboxylase (OXDC) (EC 4.1.1.2) refers to an oxalate carboxylase. Oxalate decarboxylases are a group of enzymes known in the art that catalyze the oxidation of oxalate to carbon dioxide and formate independently of molecular oxygen (O2) according to the following reaction:

HO ₂ C-CO ₂ H→1CO ₂ +HCOOH

Isoforms of oxalate decarboxylase and glycoforms of those isoforms are included in this definition. The term includes OXDCs from plants, bacteria, and fungi, including true oxalate decarboxylases from bacteria and fungi, such as Bacillus subtilis, Flammulina velutipes or Firefly, Aspergillus niger, Pseudomonas sp., Synechocystis sp., Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum, T. versicolor, Postia placenta, Myrotheca verrucosa, Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus, Reytia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovoraxparadoxus, Xanthomonas autotrophica, Aspergillus sp., Penicillium sp., and Mucor sp. Optionally, OXDC may additionally be dependent on Coenzyme A, such as OXDC from an intestinal organism. In certain instances, OXDC is a soluble hexameric protein.

Oxalate decarboxylase is produced by higher plants, bacteria, and fungi and has oxalate carboxylase activity. Oxalate decarboxylases include those produced by Bacillus subtilis, Flammulina velutipes or Fire Mushroom, Aspergillus niger, Pseudomonas sp., Synechocystis sp., Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum, T. versicolor, Postia placenta, Myrotheca verrucosa, Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus, Reytia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovorax paradoxus, Xanthomonas autotrophicus, Aspergillus sp., Penicillium sp., and Mucor sp., which are generally identified as cupin-type OXDCs. Cupin-like proteins are a large family of proteins that share certain structural features. OXDC, such as G-OXDC from the genus Collybia, is active as, for example, a hexameric glycoprotein.

The oxalate decarboxylase used to prepare crystals and for use in the methods described herein can be isolated from, for example, a natural source, or can be derived from a natural source. As used herein, the term "derived from" refers to having an amino acid or nucleic acid sequence naturally occurring in a source. For example, an oxalate decarboxylase derived from Bacillus subtilis comprises the primary sequence of a Bacillus subtilis oxalate decarboxylase protein, or is encoded by a nucleic acid comprising a sequence encoding an oxalate decarboxylase or a degradation product thereof found in Bacillus subtilis. Proteins or nucleic acids derived from a source include molecules isolated from that source, recombinantly produced, and/or chemically synthesized or modified. Crystals provided herein can be prepared from polypeptides comprising the amino acid sequence of OXDC or a functional fragment of OXDC that retains oxalate degradation activity. Preferably, OXDC retains at least one functional characteristic of naturally occurring OXDC, for example, retaining one or more of the following: the ability to catalyze oxalate degradation, the ability to polymerize, and/or the manganese requirement.

Isolated oxalate decarboxylase. Oxalate decarboxylase has been previously isolated and can be obtained from a number of sources, including Bacillus subtilis, Flammulina velutipes or Flammulina velutipes, Aspergillus niger, Pseudomonas sp., Synechocystis sp., Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum, T. versicolor, Postia placenta, Myrotheca verrucosa, Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus, Reytia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovorax paradoxus, Xanthomonas autotrophica, Aspergillus sp., Penicillium sp., and Mucor sp. OXDC can also be purchased from commercial suppliers, such as Sigma. Methods for isolating OXDC from natural sources have been described, for example, in the following references: Tanner et al., The Journal of Biological Chemistry. 47 : 43627-43634. (2001); Dashek, W. Van and Micales, JA, Methods in plant biochemistry and molecular biology. Boca Raton, FL: CRC Press. 5: 49-71. (1997); Magro et al., FEMS Microbiology Letters. 49 : 49-52. (1988); Anand et al., Biochemistry. 41 : 7659-7669. (2002); and Tanner and Bornemann, S. Journal of Bacteriology. 182 : 5271-5273 (2000). These isolated oxalate decarboxylases can be used to form the crystals and methods described herein.

Recombinant oxalate decarboxylase. Alternatively, recombinant OXDC can be used to form crystals and methods provided herein. In some cases, the recombinant OXDC comprises a sequence derived from a naturally occurring OXDC sequence, or is encoded thereby. In addition, OXDC comprising an amino acid sequence homologous to or substantially identical to a naturally occurring sequence is described herein. OXDC encoded by a nucleic acid homologous to or substantially identical to a naturally occurring OXDC-encoding nucleic acid is also provided and can be crystallized and/or administered as described herein.

As used herein, "recombinant" polypeptides are polypeptides that have been produced by recombinant DNA methods, including those produced by manipulations that rely on artificial recombination, such as polymerase chain reaction (PCR) and/or cloning into vectors using restriction enzymes. A "recombinant" polypeptide is also a polypeptide with altered expression, such as a naturally occurring polypeptide with recombinantly modified expression in a cell (e.g., a host cell).

In one embodiment, OXDC is recombinantly produced from a nucleic acid homologous to a Bacillus subtilis or Flammulina velutipes OXDC nucleic acid sequence, and is sometimes modified, for example, to enhance or optimize recombinant production in a heterologous host. An example of such a modified sequence is provided in SEQ ID NO:1 (nucleic acid), which comprises the nucleic acid sequence of the open reading frame of Flammulina velutipes OXDC, for expression in Candida boidinii. The OXDC sequence has been modified to reduce its GC content, linked to an alpha mating factor secretion signal sequence, and flanked by engineered restriction endonuclease cleavage sites. In another embodiment, OXDC is recombinantly produced from the unmodified Bacillus subtilis OXDC nucleic acid sequence of SEQ ID NO:2 or available in GenBank Accession No: Z99120. The amino acid sequence encoded by SEQ ID NO:2 is provided as SEQ ID NO:3.

OXDC polypeptides useful for forming OXDC crystals can be expressed in host cells, such as host cells containing a nucleic acid construct comprising a coding sequence for an OXDC polypeptide or a functional fragment thereof. Suitable host cells for expressing OXDC can be yeast, bacterial, fungal, insect, plant, or mammalian cells, for example, transgenic plants, transgenic animals, or cell-free systems. Preferably, the host cell is capable of glycosylation of the OXDC polypeptide, disulfide bond formation, secretion of OXDC, and/or support multimerization of the OXDC polypeptide. Preferred host cells include, but are not limited to, Escherichia coli (including E. coli Origami B and E. coli BL21), Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus subtilis, Aspergillus, Sf9 cells, Chinese hamster ovary (CHO), 293 cells (human embryonic kidney), and other human cells. Transgenic plants and transgenic animals (including pigs, cattle, goats, horses, chickens, and rabbits) are also suitable hosts for producing OXDC.

For recombinant production of OXDC, the host or host cell should contain a construct in the form of a plasmid, vector, phagemid, or transcription or expression cassette containing at least one nucleic acid encoding OXDC or a functional fragment thereof. A variety of constructs are available, including constructs maintained as a single copy or multiple copies, or constructs incorporated into the host cell chromosome. Many recombinant expression systems, components, and recombinant expression reagents are commercially available, for example, from Invitrogen Corporation (Carlsbad, CA); U.S. Biological (Swampscott, MA); BD Biosciences Pharmingen (San Diego, CA); Novagen (Madison, WI); Stratagene (La Jolla, CA); and Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH (DSMZ), (Braunschweig, Germany).

Recombinant expression of OXDC is optionally controlled by a heterologous promoter, including constitutive and/or inducible promoters. Promoters such as T7, alcohol oxidase (AOX) promoter, dihydroxy-acetone synthase (DAS) promoter, Gal1,10 promoter, phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, alcohol dehydrogenase promoter, copper metallothionein (CUP1) promoter, acid phosphatase promoter, CMV and polyhedrin promoter are also suitable. The specific promoter is selected based on the host or host cell. In addition, promoters inducible by methanol, copper sulfate, galactose, low phosphate, alcohol (e.g., ethanol) can also be used and are well known in the art.

The nucleic acid encoding OXDC may optionally include heterologous sequences. For example, in some embodiments, a secretory sequence is included at the N-terminus of the OXDC polypeptide. Signal sequences such as those from α-mating factor, BGL2, yeast acid phosphatase (PHO), xylanase, α-amylase, those from other yeast secreted proteins, and secretory signal peptides derived from other species that can direct secretion by host cells may be used. Similarly, other heterologous sequences such as linkers (e.g., comprising cleavage or restriction endonuclease sites) and one or more expression control elements, enhancers, terminators, leader sequences, and one or more translation signals are within the scope of this description. These sequences may optionally be included in the construct and/or linked to the nucleic acid encoding OXDC. Unless otherwise indicated, "linked" sequences may be directly or indirectly bound to one another.

Similarly, epitope or affinity tags such as histidine, HA (hemagglutinin peptide), maltose binding protein, FLAG or glutathione-S-transferase can be optionally attached to the OXDC polypeptide. After production or purification, the tag can be optionally cleaved from the OXDC. A skilled artisan can readily select an appropriate heterologous sequence, for example, one that matches the host cell, construct, promoter, and/or secretion signal sequence.

OXDC homologs or variants differ from the OXDC reference sequence by one or more residues. For example, some designated amino acids may be substituted with structurally similar amino acids. Structurally similar amino acids include: (I, L, and V); (F and Y); (K and R); (Q and N); (D and E); and (G and A). OXDC homologs described herein also include deletions, additions, or substitutions of amino acids. Such homologs and variants include: (i) polymorphic variants and natural or artificial mutants, (ii) modified polypeptides in which one or more residues are modified, and (iii) mutants comprising one or more modified residues.

An OXDC polypeptide or nucleic acid is "homologous" (or a "homolog") if it is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the reference sequence. A homolog is a "variant" if it differs from the reference sequence in nucleotide or amino acid sequence by no more than 1, 2, 3, 4, 5, 8, 10, 20, or 50 residues and retains the ability to catalyze oxalate degradation (or encodes a polypeptide that retains this ability). Fragments of oxalate decarboxylase can be homologs, including variants and/or substantially identical sequences. As an example, homologs can be derived from different OXDC sources, or they can be derived from or related to a reference sequence by truncation, deletion, substitution, or addition mutations. The percent identity between two nucleotide or amino acid sequences can be determined by a standard alignment algorithm, for example, the Basic Local Alignment Tool (BLAST) described in Altschul et al., J. Mol. Biol., 215 : 403-410 (1990), the algorithm of Needleman et al., J. Mol. Biol., 48 : 444-453 (1970), or the algorithm of Meyers et al., Comput. Appl. Biosci. 4 : 1117 (1988). Such algorithms are incorporated into BLASTN, BLASTP, and "BLAST2Sequences" programs (for review, see McGinnis and Madden, Nucleic Acids Res. 32 : W20-W25, 2004). When using such programs, the default parameters can be used. For example, for nucleotide sequences, the following settings can be used for "BLAST2Sequences": program BLASTN, reward for match 2, penalty for mismatch 2, open gap and extension gap penalties of 5 and 2, respectively, gap x_dropoff 50, expect 10, word size 11, filter ON. For amino acid sequences, the following settings can be used for "BLAST2Sequences": program BLASTP, matrix BLOSUM62, open gap and extension gap penalties of 11 and 1, respectively, gap x_dropoff 50, expect 10, word size 3, filter ON. Amino acid and nucleic acid sequences of OXDC suitable for forming crystals described herein can include homologous, variant, or substantially identical sequences.

Purification of oxalate decarboxylase. Prior to crystallization, the oxalate decarboxylase protein or polypeptide can be purified from a source (e.g., a natural or recombinant source). "Isolated" polypeptides herein are polypeptides that have been substantially separated from their natural environment (e.g., their origin (e.g., cells, proteins, lipids, and/or nucleic acids of tissues (i.e., plant tissues), or liquid or culture medium (in the case of secreted polypeptides)). Isolated polypeptides include those obtained by the methods described herein or other suitable methods, including substantially pure or substantially pure polypeptides, and polypeptides produced by chemical synthesis, recombinant production, or a combination of biological and chemical methods. Optionally, the isolated protein is further processed after production, such as by a purification step.

Purification can include buffer exchange and chromatography steps. Optionally, a concentration step can be used, for example, by dialysis, chromatofocusing chromatography, and/or in combination with buffer exchange. In some cases, cation or anion exchange chromatography is used for purification, including Q-agarose, DEAE agarose, DE52, sulfopropyl agarose chromatography or CM52 or similar cation exchange columns. Buffer exchange is optionally performed prior to chromatographic separation and can be achieved by tangential flow filtration, such as diafiltration. In certain preparations, OXDC is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% pure.

Purification performed on a gram scale is suitable for preparing OXDC and optimizes the process for efficient, inexpensive, production-scale purification of OXDC. For example, purification of at least 0.5, 1, 2, 5, 10, 20, 50, 100, 500, or 1000 grams or more of OXDC is provided in a purification process. In an exemplary process, tangential flow filtration of at least 10 L, 50 L, 100 L, 500 L, 1000 L, or more of a starting sample is provided to achieve buffer exchange and precipitation of contaminating proteins. A single Q-Sepharose column is optionally used to purify OXDC.

Crystallization of purified OXDC can also remove contaminants, e.g., to further purify an OXDC preparation. For example, compared to soluble purified OXDC, crystallized OXDC, as described in Examples 2-6, has reduced levels of low molecular weight contaminants. In some aspects, contaminants having a measured mass of 0-10 KDa, 1-10 KDa, 0.5-5 KDa, or 2-5 KDa (as analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)) are selectively excluded from the crystalline form. For example, contaminants having a measured mass of approximately 2.5, 3.0, 3.7, 3.8, 4.0, 4.2, or 5.0 KDa can be substantially removed by crystallization. Crystallization purification can also be performed using, for example, fermentation medium containing crude oxalate decarboxylase.

Crystallization of oxalate decarboxylase. Using the above-mentioned OXDC polypeptide, for example, a hexamer, oxalate decarboxylase crystals can be prepared (see Anand et al., Biochemistry 41 : 7659-7669 (2002)). For example, vapor diffusion (e.g., hanging drop and sitting drop methods) and batch crystallization methods can be used. Oxalate decarboxylase crystals can be gradually formed by controlled crystallization of the protein from an aqueous solution or an aqueous solution containing an organic solvent. Controllable conditions include, for example, the evaporation rate of the solvent, the presence of appropriate cosolutes and buffers, pH, and temperature.

For therapeutic applications, such as for treating conditions or disorders associated with oxalate levels, a variety of OXDC crystal sizes are suitable. In certain embodiments, crystals having an average size of less than about 500 μm are used. Oxalate decarboxylase crystals having an average, maximum, or minimum size (e.g., of about 0.01, 0.1, 1, 5, 10, 25, 50, 100, 200, 300, 400, 500, or 1000 μm in length are also provided. Microcrystalline showers are also suitable.

Ranges are suitable and will be appreciated by those skilled in the art. For example, protein crystals may have a longest dimension ranging from about 0.01 μm to about 500 μm, or from 0.1 μm to about 50 μm. In a specific embodiment, the longest dimension ranges from about 0.1 μm to about 10 μm. Crystals may also have a shape selected from spheres, needles, rods, plates, such as hexagons and squares, rhombuses, cubes, bipyramids, and prisms. In illustrative embodiments, the crystals are cubes having a longest dimension less than 5 μm.

In general, crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or an aqueous solvent containing an appropriate crystallizing agent (e.g., salt or organic solvent). The solvent is combined with the protein and optionally stirred at a temperature suitable for inducing crystallization and maintaining acceptable protein activity and stability in experimental determinations. The solvent may optionally include cosolutes, such as monovalent or divalent cations, cofactors or chaotropic agents, and buffers for controlling pH. Experimental determinations promote the cosolutes and their concentrations required for crystallization. In industrial-scale processes, controlled precipitation of crystals can be performed by combining, for example, protein, precipitant, cosolute, and optional buffer in a batch process. Alternative laboratory crystallization methods and conditions can be adopted, such as dialysis or vapor diffusion (McPherson, et al., Methods Enzymol. 114 : 112-20 (1985) and Gilliland, Crystal Growth 90 : 51-59 (1998)). Occasionally, incompatibility between the cross-linking agent and the crystallization medium may require changing the buffer (solvent) before cross-linking.

As described in the Examples, oxalate decarboxylase crystallizes under a variety of conditions, including a wide pH range (e.g., pH 3.5-8.0). In some of the embodiments described, a precipitant such as polyethylene glycol (e.g., PEG 200, PEG 400, PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 8000 [see Examples 7 and 8]) or an organic cosolvent such as 2-methyl-2,4-pentanediol (MPD) is included. Common salts that can be used include sodium chloride, potassium chloride, ammonium sulfate, zinc acetate, and the like.

The concentration of oxalate decarboxylase in the crystallization fermentation broth can be, for example, at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90, or 100mg/ml, or higher. The efficiency or productive rate of crystallization reaction are at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In one embodiment, oxalate decarboxylase solution is mixed with suitable buffer by batch process, gradually form or produce oxalate decarboxylase crystals. In certain embodiments, the buffer is 100mMTris-HCl buffer pH 8.0 and 100mMNaCl containing 2mM cysteine-HCl.

Crystallization from cells or cell extracts. Crystals can be prepared directly from cells or crude cell extracts. In one embodiment, bacterial cells expressing oxalate decarboxylase are harvested. Cells are resuspended in the presence or absence of DNA enzyme and homogenized. A salt solution is added to the cell lysate to a salt concentration of about 0.3M, 0.4M, 0.5M, 0.6M or higher. The added salt can be a sodium salt, potassium salt, calcium salt or other salt. By removing cell debris, protein can be optionally extracted from the cell mixture. In one embodiment, the homogenized cell mixture is centrifuged so that the protein is in the supernatant solution. By reducing the salt concentration of the cell mixture or protein solution, crystals are produced. In one embodiment, salt is removed by dialysis to maintain protein concentration. In order to improve the crystal yield, the protein solution can be concentrated before reducing the salt concentration of the solution. Crystals can be produced in a solution of about pH 6, 7 or 8.

Crystals can be prepared from protein precipitates or particles. In one embodiment, cells expressing the target protein are harvested and the oxalate decarboxylase protein is collected in the precipitate or particles. The particles or precipitates containing the oxalate decarboxylase protein are dissolved in a saline solution. Crystals are formed by reducing the salt concentration of the protein solution. In order to improve the crystal yield, the salt concentration in the dissolved protein solution is at least about 0.3M, 0.4M, 0.5M or higher before reducing the concentration to produce crystals.

Crystals can also be prepared from protein solutions. In one embodiment, a solution of oxalate decarboxylase protein is concentrated in a saline solution, and crystals are formed when the salt concentration of the solution is reduced. To increase crystal yield, the salt concentration is at least about 0.3 M, 0.4 M, 0.5 M, or higher before reducing the concentration to produce crystals.

Stabilized crystals. Once oxalate decarboxylase crystals have gradually formed in a suitable medium, they can optionally be stabilized, for example by cross-linking. Cross-linking results in stabilization of the crystal lattice by introducing covalent bonds between the component protein molecules of the crystal. This makes it possible to transfer the protein to an alternative environment that might otherwise be incompatible with the presence of the crystal lattice or even incompatible with the presence of the intact protein. Oxalate decarboxylase crystals can be cross-linked by, for example, lysine amine groups, sulfhydryl groups (sulfhydryl groups), and carbohydrate moieties. Cross-linked crystals are also referred to herein as "OXDC-CLEC,""CLEC-OXDC," or "CLEC."

Cross-linked crystals can alter enzyme stability (e.g., pH, temperature, mechanical and/or chemical stability), pH properties of OXDC activity, solubility, uniformity of crystal size or volume, rate of enzyme release from the crystals, and/or the size and shape of pores between individual enzyme molecules in the underlying crystalline lattice.

Advantageously, crosslinking or stabilization according to the present invention is performed in a manner such that the crystals comprise OXDC that exhibit at least 60%, 80%, 100%, 150%, 200%, 250%, 300% or more activity compared to soluble OXDC. Stability can be improved by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more compared to soluble OXDC. Stability can be measured under storage conditions, such as pH stability, temperature stability, stability against intestinal proteases, dissolution stability, and in vivo biostability, for example.

In some embodiments, crosslinking slows the dissolution of the OXDC polypeptide from the crystals into solution, effectively immobilizing the protein molecule within the microcrystalline particles. Upon exposure to a trigger in the environment surrounding the crosslinked protein crystals, e.g., under use conditions rather than storage conditions, the protein molecule slowly dissolves, releasing active OXDC polypeptide and/or increasing OXDC activity. The dissolution rate can be controlled, for example, by one or more of the following factors: the degree of crosslinking, the length of time the protein crystals are exposed to the crosslinking agent, the rate at which the crosslinking agent is added to the protein crystals, the nature of the crosslinking agent, the chain length of the crosslinking agent, pH, temperature, the presence of sulfahydryl reagents (e.g., cysteine, glutathione), the surface area of the crosslinked protein crystals, the size of the crosslinked protein crystals, and the shape of the crosslinked protein crystals.

Cross-linking can be achieved using one or a combination of multiple cross-linking agents simultaneously (in parallel) or sequentially, including multifunctional agents. After exposure to a trigger in the surrounding environment, or after a given time period, the cross-linking between the protein crystals cross-linked with such a multifunctional cross-linking agent becomes smaller or weaker, resulting in protein dissolution or activity release. Alternatively, the cross-linking can be broken at the binding point, resulting in protein dissolution or activity release. See U.S. Patent Nos. 5,976,529 and 6,140,475.

In some embodiments, the cross-linking agent is a multifunctional cross-linking agent having at least 2, 3, 4, 5, or more reactive moieties. In various embodiments, the agent can be selected from glutaraldehyde, succinyldialdehyde, suberaldehyde, glyoxal, dithiobis(succinimidyl propionate), 3,3'-dithiobis(sulfosuccinimidyl propionate), 3,3'-dithiobispropionimidate dimethyl HCl, N-succinimidyl-3-(2-pyridyldithio)propionate, cyclohexanediamine, diaminooctane, ethylenediamine, succinic anhydride, phenylglutaric anhydride, salicylaldehyde, acetimidate, formalin, acrolein, succinic semialdehyde, butyraldehyde, lauryl aldehyde, glyceraldehyde, and trans-oct-2-enal.

Other multifunctional crosslinking agents include halogenated triazines, such as cyanuric chloride; halogenated pyrimidines, such as 2,4,6-trichloro/bromopyrimidine; anhydrides or halides of aliphatic or aromatic mono- or dicarboxylic acids, such as maleic anhydride, (meth)acryloyl chloride, chloroacetyl chloride; N-methylol compounds, such as N-methylol chloro-acetamide; diisocyanates or diisothiocyanates, such as phenylene-1,4-diisocyanate and aziridine. Other crosslinking agents include epoxides, such as diepoxides, triepoxides, and tetraepoxides. In one embodiment, the crosslinking agent is glutaraldehyde, a bifunctional agent, used alone or sequentially with an epoxide. Other crosslinking agents may also be used simultaneously (in parallel) or sequentially with reversible crosslinkers, such as those described below (see, for example, the 1996 catalog of Pierce Chemical Company).

According to an alternative embodiment of the present invention, crosslinking can be performed in parallel or sequentially using reversible crosslinkers. The resulting crosslinked protein crystals are characterized by reactive multifunctional crosslinkers in which a trigger is incorporated as a spacer group. The reactive functional group is involved in linking the reactive amino acid side chains in the protein together, and the trigger consists of a bond that can be broken by changing one or more conditions in the surrounding environment (e.g., pH, the presence of a reducing agent, temperature, or thermodynamic water activity).

The crosslinking agent may be homofunctional or heterofunctional. The reactive functional group (or moiety) may, for example, be selected from one of the following functional groups (wherein R, R', R", and R''' may be an alkyl, aryl or hydrogen group):

I. Reactive acyl donors, for example: carboxylates RCOOR', amides RCONHR', acyl azides RCON ₃ , carbodiimides RN=C=N-R', N-hydroxy iminoesters, RCO-O-NR', iminoesters RC=NH2 ⁺ (OR'), anhydrides RCO-O-COR', carbonates RO-CO-O-R', urethanes RNHCONHR', acid halides RCOHal (wherein Hal=halogen), acylhydrazines RCONNR'R", and (O-acylisoureas) OAcylisoureas RCO-OC=NR'(-NR"R''');

II. Reactive carbonyl groups, for example: aldehydes RCHO and ketones RCOR', acetals RCO(H ₂ )R', and ketals RR'CO2R'R" (functional groups containing reactive carbonyl groups known to those skilled in the art of protein immobilization and cross-linking are described in the literature (Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, 111. (1994); SS Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla. (1991));

III. Alkyl or aryl donors, for example: alkyl or aryl halides R-Hal, azides RN ₃ , sulfates RSO ₃ R', phosphates RPO(OR' ₃ ), alkyloxoniums R ₃ O+, sulfoniums R ₃ S+, nitrates RONO ₂ , Michael acceptors RCR'=CR'''COR", aryl fluorides ArF, isonitriles RN+=C-, haloamines R ₂ N-Hal, alkenes, and alkynes;

IV. sulfur-containing groups, such as: disulfide RSSR ', thiol RSH, and epoxide R ₂ C_ ^O CR '₂; and

V. Salts, for example: alkyl or aryl ammonium salts R ₄ N + , carboxylates RCOO- , sulfates ROSO ₃ - , phosphates ROPO ₃ ″ , and amines R ₃ N.

Reversible cross-linking agents, for example, include triggers. Triggers include alkyl, aryl, or other chains with activated groups that react with the protein to be cross-linked. Those reactive groups can be any type of group, such as those susceptible to nucleophilic displacement, free radical displacement, or electrophilic displacement, including halides, aldehydes, carbonates, urethanes, xanthane, and epoxides, among others. For example, the reactive group can be unstable to acids, bases, fluorides, enzymes, reduction, oxidation, sulfhydryls, metals, photolysis, radicals, or heat.

Other examples of reversible cross-linking agents are described in TW Green, Protective Groups in Organic Synthesis, John Wiley & Sons (Eds.) (1981). Any variety of strategies for reversible protecting groups can be incorporated into cross-linking agents suitable for producing cross-linked protein crystals that can achieve reversible controlled dissolution. Various protocols are listed in Waldmann's Angewante Chemie Inl. Ed. Engl., 35 : 2056 (1996) in a review of this topic.

Another type of reversible cross-linking agent is a disulfide cross-linking agent. The trigger for disruption of cross-link formation by such a cross-linking agent is the addition of a reducing agent, such as cysteine, to the environment of the cross-linked protein crystal. Exemplary disulfide cross-linking agents are described in the Pierce Catalog and Handbook (1994-1995). Examples of such cross-linking agents and methods are disclosed in U.S. Patent No. 6,541,606, the relevant portions of which are incorporated by reference.

Additionally, cross-linking agents that cross-link between carbohydrate moieties or between carbohydrate moieties and amino acids can also be used.

The concentration of the cross-linking agent solution can be about 0.01% to 20%, about 0.02% to 10%, or about 0.05% to 5% w/v. Typically, the cross-linking agent is about 0.5% or about 1% w/v. For example, the concentration of the cross-linking agent solution can be, for example, about 0.01%, 0.02%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% w/v [see Table 2 in the Examples]. It may be necessary to change the buffer solution before cross-linking. Crystals, including CLEC, can be optionally lyophilized or otherwise prepared.

Crystals, including cross-linked crystals described herein, can be used in the methods of treatment and methods of reducing oxalate levels described herein. OXDC crystals can also be used in methods related to industrial processes (e.g., synthesis, processing, bioremediation, disinfection, sterilization), and methods of treating plants (e.g., fungal infections in plants), as reviewed, for example, in Svedruzic et al., Arch. Biochem. Biophys. 433 : 176-192 (2005). Such non-therapeutic uses of soluble or amorphous OXDC are described, for example, in U.S. Patent Nos. 5,866,778; 6,218,134; 6,229,065; 6,235,530; and 6,503,507. Based on one or more properties of the stabilized OXDC crystals described above, the crystals described herein can be used for these applications, such as increasing the stability of oxalate decarboxylase.

The drying of oxalate decarboxylase crystals . Remove water, organic solvent or liquid polymer in the following manner, thereby dry oxalate decarboxylase crystals: comprising, with nitrogen, air or inert gas drying, vacuum oven drying, lyophilization, washing with volatile organic solvent, evaporating solvent subsequently, evaporation in a fume hood, tray drying, fluidized bed drying, spray drying, vacuum drying, or drum drying. Usually, when crystal becomes free-flowing powder, dry. By making a gas stream pass through the wet crystal, dry. The gas can be selected from: nitrogen, argon, helium, carbon dioxide, air or their combination.

In principle, dry crystals can be prepared by lyophilization. However, this technique involves rapid cooling of the substance and is only suitable for freeze-drying stable products. In one embodiment, an aqueous solution containing crystalline oxalate decarboxylase is first frozen to -40 to -50°C and then removed under vacuum.

Production of oxalate decarboxylase crystals or preparations or compositions comprising such crystals. In one aspect, oxalate decarboxylase crystals or preparations or compositions comprising such crystals are disclosed. Such compositions can be prepared according to the following method:

First, oxalate decarboxylase is crystallized. Next, an excipient or component selected from a sugar, sugar alcohol, tackifier, wetting agent or solubilizing agent, buffer salt, emulsifier, antimicrobial agent, antioxidant, and coating agent is directly added to the mother liquor. Alternatively, the mother liquor is removed and the crystals are suspended in the excipient solution for a minimum of 1 hour to a maximum of 24 hours. The excipient concentration is typically about 0.01 to about 10% (w/w). The component concentration is about 0.01 to about 90% (w/w). The crystal concentration is about 0.01 to about 99% (w/w).

The mother liquor is then removed from the crystal slurry by filtration or by centrifugation. The crystals are then optionally washed with about 50-100% (w/w) solution of one or more organic solvents (e.g., ethanol, methanol, isopropanol, or ethyl acetate) at room temperature or at a temperature of about -20°C to about 25°C.

The crystals are then dried by passing nitrogen, air, or an inert gas through them. Alternatively, the crystals can be dried by air drying, spray drying, lyophilization, or vacuum drying. After washing, the crystals are dried for a minimum of about 1 hour and a maximum of about 72 hours until the water content of the final product is less than about 10% by weight, more preferably less than about 5% by weight. Finally, the crystals can be micronized (reduced in size) if desired.

According to one embodiment of the present invention, when preparing oxalate decarboxylase crystals or a preparation or composition comprising such crystals, no reinforcing agent, such as a surfactant, is added during the crystallization process. After crystallization, the excipient or ingredient is added to the mother liquor at a concentration of about 1 to about 10% (w/w), or at a concentration of about 0.1 to about 25% (w/w), or at a concentration of about 0.1 to about 50% (w/w). The excipient or ingredient is incubated with the crystals in the mother liquor for about 0.1 to about 3 hours, or for about 0.1 to about 12 hours, or for about 0.1 to about 24 hours.

In another embodiment of the present invention, the ingredients or excipients are dissolved in a solution other than the mother liquor, the crystals are taken out from the mother liquor, and suspended in the excipient or ingredient solution. The ingredient or excipient concentrations and incubation times are the same as those described above.

Another advantage of the present invention is that the oxalate decarboxylase crystals or their formulations encapsulated in the polymer carrier can be dried by lyophilization to form a composition comprising microspheres. Lyophilization or freeze drying allows water to be separated from the composition. The oxalate decarboxylase crystalline composition is first frozen and then placed in a high vacuum. In the vacuum, the water of crystallization sublimes, leaving the oxalate decarboxylase crystalline composition containing only tightly bound water. Such processing further stabilizes the composition and makes storage and transportation at common ambient temperatures easier.

Spray drying allows water to be separated from crystalline products. It is ideally suited for the continuous production of dry solids in the form of powders, granules, or agglomerates from liquid feedstocks in the form of solutions, emulsions, and pumpable suspensions. Spray drying involves atomizing the liquid feedstock into a spray of fine droplets and exposing the droplets to hot air within a drying chamber. The spray is generated by a rotary (wheel) or nozzle atomizer. Under controlled temperature and airflow conditions, water evaporates from the droplets, forming dry particles. Spray drying requires relatively high temperatures. However, thermal damage to the product is usually only slight due to the evaporative cooling effect during the critical drying stage and because the subsequent exposure of the dried material to high temperatures is very short. The powder is continuously discharged from the drying chamber. The operating conditions and dryer design are selected based on the drying characteristics of the product and the powder specifications. Spray drying is an ideal process where the final product must meet precise quality standards in terms of particle size distribution, residual water content, bulk density, and particle shape.

Compositions. OXDC crystals, including cross-linked crystals, are provided as compositions, such as pharmaceutical compositions (see, for example, U.S. Patent No. 6,541,606, which describes formulations and compositions of protein crystals). Pharmaceutical compositions containing OXDC crystals include OXDC crystals and one or more ingredients or excipients, including, but not limited to, sugars and biocompatible polymers. Examples of excipients are described in the Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain, and other examples are described below.

OXDC enzymes can be administered as crystals in a composition as any of a variety of physiologically acceptable salt forms and/or as part of a pharmaceutical composition with acceptable pharmaceutical carriers and/or additives. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to those skilled in the art (see, for example, Physician's Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, ed. Gennado et al., 20th ed., Lippincott, Williams & Wilkins, 2000). For the purposes of this application, "formulation" includes "crystalline formulation".

The oxalate decarboxylase that can be used in the inventive method can be combined with an excipient. According to the present invention, an "excipient" is used as a filler or a filler combination used in a pharmaceutical composition. Exemplary ingredients and excipients used in the composition are described below.

Biocompatible polymers . Biocompatible polymers are polymers that are non-antigenic (when not used as adjuvants), non-carcinogenic, non-toxic, and otherwise not inherently incompatible with living organisms and can be used in the OXDC crystal compositions described herein. Examples include: poly(acrylic acid), poly(cyanoacrylate), poly(amino acids), poly(anhydrides), poly(depsipeptides), poly(esters) such as poly(lactic acid) or PLA, poly(lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutyrate), poly(caprolactone) and poly(dioxanone); poly(ethylene glycol), poly((hydroxypropyl)isobutylmethacrylamide, poly[(organo)phosphazene], poly(o-esters), poly(vinyl alcohol), poly(vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginates, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycosaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

Biodegradable polymers , i.e., polymers that can be degraded by hydrolysis or dissolution, can be included in the OXDC crystal compositions. Degradation can be heterogeneous (occurring primarily at the particle surface) or homogeneous (uniform degradation throughout the polymer matrix).

Ingredients such as one or more excipients or pharmaceutical ingredients or excipients may be included in the OXDC crystal composition. The ingredients may be inert or active ingredients.

Methods for treating oxalate-related conditions using OXDC crystals . The methods of the present invention comprise administering oxalate decarboxylase, such as OXDC crystals or a cross-linked form thereof, to a mammalian subject to treat, prevent, or reduce the risk of a condition associated with elevated oxalate levels. Elevated oxalate levels can be detected, for example, in a biological sample from the subject, such as a body fluid, including urine, blood, serum, or plasma. In certain embodiments, urine oxalate levels are detected. In the methods described herein, crystals and/or compositions comprising crystals can be administered.

In some embodiments, methods are provided for treating hyperoxaluria in individuals with primary hyperoxaluria, enteric hyperoxaluria, surgically induced hyperoxaluria, idiopathic hyperoxaluria, and oxalate deposition. In other cases, elevated oxalate-related conditions of the kidneys, bones, liver, gastrointestinal tract, and pancreas are amenable to treatment as disclosed herein. Other conditions or diseases treated by the methods provided herein include, but are not limited to, ethylene glycol (oxalate) poisoning, idiopathic urolithiasis, renal failure (including progressive, chronic, or end-stage renal failure), steatorrhea, malabsorption, ileopathy, vulvodynia, cardiac conduction disorders, inflammatory bowel disease, cystic fibrosis, pancreatic exocrine insufficiency, Crohn's disease, ulcerative colitis, nephrocalcinosis, osteoporosis, urolithiasis, and kidney stones. Such conditions and disorders may optionally be acute or chronic.

The methods of the present invention can reduce oxalate levels in a subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to levels in untreated or control subjects. In some embodiments, the reduction is measured by comparing the subject's oxalate levels before and after administration of OXDC. In some embodiments, the present invention provides methods for treating or ameliorating an oxalate-related condition or disorder such that one or more symptoms of the condition or disorder are increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In certain embodiments, the methods reduce endogenous oxalate levels and/or dietary oxalate absorption.

In some embodiments, methods are provided for treating individuals with a genotype associated with high oxalate levels, such as individuals homozygous or heterozygous for a mutation that reduces the activity of, for example, alanine:glyoxylate aminotransferase, glyoxylate reductase/hydroxypyruvate reductase, hepatic glycolate oxidase, or another enzyme involved in oxalate metabolism or associated with hyperoxaluria. In other embodiments, methods are provided for treating individuals with reduced or absent intestinal colonization of Oxalobacter formigenes.

The disclosed method comprises administering a therapeutically effective amount of oxalate decarboxylase to a mammalian subject susceptible to, suffering from, or at risk of developing a condition associated with elevated oxalate levels. The populations treatable by the methods of the present invention include, but are not limited to, subjects suffering from or at risk of developing an oxalate-related condition (e.g., primary hyperoxaluria or enteric hyperoxaluria).

Subjects treated according to the methods of the present invention include, but are not limited to, mammals, including humans, non-human primates, primates, baboons, gorillas, monkeys, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

Indications, Symptoms, and Disease Indications. A variety of methods can be used to assess the development or progression of oxalate-related conditions or conditions associated with elevated oxalate levels. Such conditions include, but are not limited to, any condition, disease, or disorder as defined above. The development or progression of an oxalate-related condition can be assessed, for example, by measuring urine oxalate, plasma oxalate, measuring renal or liver function, or detecting calcium oxalate deposits.

By detecting or measuring the oxalate concentration in, for example, a urine sample or other biological sample or liquid, it is possible to identify a disease, disease or obstacle. The early symptoms of hyperoxaluria are typically kidney stones, which may be accompanied by severe or sudden abdominal pain or flank pain, hematuria, frequent urination, pain during urination or fever and chills. Kidney stones can be symptomatic or asymptomatic and can be observed by, for example, abdominal X-rays, ultrasound or computed tomography (CT) scanning imaging. If hyperoxaluria is not controlled, the kidney will be damaged, and renal function will be impaired. The kidney may even fail. By the reduction or lack of urine output (glomerular filtration rate), general feeling of illness, tiredness and significant fatigue, nausea, vomiting, anemia and/or being difficult to develop and grow normally in early childhood, it is possible to identify renal failure (and low renal function). By methods such as direct visual inspection (e.g., with the eyes), X-rays, ultrasound, CT, echocardiography or biopsy (e.g., bone, liver or kidney), it is also possible to detect calcium oxalate deposits in other tissues and organs.

Renal and liver function, as well as oxalate concentrations, can also be assessed using direct and indirect assays recognized in the art. Compound levels or urine, blood, or other biological samples can also be tested using well-known techniques. For example, oxalate, glycolate, and glycerate levels can be measured. Assays for liver and kidney function are well known, for example, analyzing liver tissue for enzyme deficiencies and analyzing kidney tissue for oxalate deposits. Samples can also be tested for DNA changes known to cause primary hyperoxaluria.

Other indications for treatment include, but are not limited to, the presence of one or more risk factors, including those discussed above and in the following sections. By ascertaining the presence or absence of one or more such risk factors, diagnostic or prognostic indicators, subjects at risk for developing or being susceptible to a condition, disease or disorder, or subjects who may be particularly susceptible to oxalate decarboxylase treatment, can be identified. Similarly, by analyzing one or more genotypic or phenotypic markers, individuals at risk for developing an oxalate-related condition can be identified.

The disclosed method can be used for subjects whose urine oxalate level per 24 hour period is at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 mg oxalate or more. In certain embodiments, oxalate level is associated with one or more symptoms or conditions. Oxalate levels can be measured in biological samples, such as body fluids, including blood, serum, plasma or urine. Optionally, oxalate is standardized to a standard protein or substance, such as creatinine in urine. In some embodiments, the claimed methods comprise administering oxalate decarboxylase to reduce circulating oxalate levels in a subject to undetectable levels, or to less than 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the subject's pre-treatment oxalate level within 1, 3, 5, 7, 9, 12, or 15 days.

Hyperoxaluria in humans is characterized by urinary oxalate excretion greater than 40 mg (about 440 μmol) or 30 mg per day. Exemplary clinical cutoff levels are, for example, 43 mg/day (about 475 μmol) for men and 32 mg/day (about 350 μmol) for women. Hyperoxaluria can also be defined as urinary oxalate excretion greater than 30 mg per gram of urine creatinine per day. Patients with mild hyperoxaluria may excrete at least 30-60 (342-684 μmol) or 40-60 (456-684 μmol) mg of oxalate per day. Patients with enteric hyperoxaluria may excrete at least 80 mg of urinary oxalate (912 μmol) per day, and patients with primary hyperoxaluria may excrete at least 200 mg (2280 μmol) per day, e.g., Borowski A.E, Langman CB. Hyperoxaluria and Oxalosis: Current Therapy and Future directions. Exp Opinion Phrama (2006, in press).

Administration of OXDC crystals and compositions thereof

Administration of oxalate decarboxylase according to the methods of the present invention is not limited to any particular delivery system, including administration through the upper gastrointestinal tract, such as by mouth (e.g., in a capsule, suspension, tablet, or with food) or the upper stomach or intestines (e.g., by catheter or injection), to reduce oxalate levels in an individual. In some cases, OXDC is administered to reduce endogenous oxalate levels and/or concentrations. OXDC can also be provided by an in vitro device, such as a dialysis device, a catheter, or a structure or device that contacts a biological sample from an individual.

Individual administration can occur in a single dose or repeated administration, and in any of a variety of physiologically acceptable forms, and/or using acceptable pharmaceutical carriers and/or additives as part of a pharmaceutical composition (described above). In the methods disclosed herein, oxalate decarboxylase can be administered alone, simultaneously, or continuously with one or more other bioactive agents, such as pyridoxine (vitamin B-6), orthophosphate, magnesium, glycosaminoglycans, calcium, iron, aluminum, magnesium, potassium citrate, cholestyramine, organic marine hydrocolloids, plant juices, such as banana stem juice or beet juice, or L-cysteine. Bioactive agents that reduce oxalate levels or increase the activity or availability of OXDC are provided. In continuous administration, oxalate decarboxylase and one or more other agents can be administered in any order. In some embodiments, the length of the overlapping intervals can be more than 2, 4, 6, 12, 24, or 48 weeks or more.

Oxalate decarboxylase can be administered as the sole active compound or in combination with another active compound or composition.Unless otherwise indicated, oxalate decarboxylase is administered at a dose of about 10 μg/kg to 25 mg/kg or 100 mg/kg, depending on the severity of symptoms and progression of the disease. A suitable therapeutically effective dose of OXDC is selected by the treating clinician and generally ranges from 10 μg/kg to 20 mg/kg, 10 μg/kg to 10 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg/kg to 1 mg/kg, 100 μg/kg to 10 mg/kg, 500 μg/kg to 5 mg/kg, 500 μg/kg to 20 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 25 mg/kg, 5 mg/kg to 100 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 25 mg/kg, and 10 mg/kg to 25 mg/kg. In addition, the specific doses indicated in the Examples or in Physician's Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002 may be used.

The oxalate decarboxylase crystals of the present invention can be administered, for example, via an extracorporeal device or catheter that delivers oxalate decarboxylase to a patient. A catheter, for example, a urinary catheter, can be coated with a composition containing oxalate decarboxylase crystals.

The following examples provide illustrative embodiments of the present invention. Those skilled in the art will appreciate that many modifications and variations can be made without altering the spirit or scope of the present invention. Such modifications and variations are within the scope of the present invention. The examples are not intended to limit the present invention in any way.

Example

Example 1. Fermentation and purification of oxalate decarboxylase.

Oxalate decarboxylase (OXDC) from Bacillus subtilis is a 261 kDa homohexameric protein composed of six identical monomers. Each monomer contains 385 amino acids, with a calculated molecular weight of ~43-44 kDa and an isoelectric point of 5.2. The OXDC gene, previously referred to as yvrK, was amplified by PCR using Bacillus subtilis genomic DNA as a template.

The amplified OXDC gene was first cloned into the pCRII vector (Invitrogen, Carlsbad, CA), then subcloned and expressed from the pET-11a expression vector using E. coli BL21(DE3)pLysS cells. This gene expression in the pET-11a vector is under the control of the T7 promoter, and OXDC expression is regulated by induction with IPTG (isopropyl-β-D-thiogalactopyranoside).

Fermentation was used to achieve high-level expression of recombinant OXDC in E. coli. Expression was performed in 800 L of fermentation medium containing casein hydrolysate (USB Corporation, Cleveland, OH) or soy peptone, yeast extract (USB Corporation), NaCl (Fisher Scientific), PPG2000 defoamer (PPG), KOH (Mallinckrodt Baker, Inc., Phillipsburg, NJ), and ampicillin (USB Corporation). Because OXDC is a manganese-dependent enzyme, 5 mM _MnCl₂ · _4H₂O (Mallinckrodt Baker, Inc.) was included in the fermentation medium. OXDC expression was induced by the addition of 0.4 mM IPTG (Lab Scientific). OXDC-expressing cells were grown in shake flasks or fermentors. Using this method, OXDC was primarily expressed in the pellet preparation.

The glycerol stock of the BL21 cell transformed with pET11a-OXDC is used for fermentation. For 800L fermentation, 2x250ml flasks are used to prepare pre-seed culture, each flask containing 50ml substratum (LB+100 μg/ml ampicillin). 2x0.5ml inoculum is used. Culture was cultivated at 35°C, 250rpm for 6 hours. The culture was then transferred to 6x2L flasks, each containing 1L substratum (LB+100 μg/ml ampicillin). In each flask, 10ml starting culture was added. These cultures were cultivated at 35°C, 250rpm for 12 hours. This culture of 6L is transferred to a fermentor tank containing the above-mentioned appropriate substratum in 800L. Culture growth is at 37°C, pH 7.0, and shaking at 100rpm makes dissolved oxygen about 30-40, until OD ₆₀₀ reaches about 0.3 (this process requires about 2-3 hours). OXDC expression was induced with 0.4 mM IPTG + 5 mM MnCl ₂ ·4H ₂ O. The culture was induced at 37°C, 100 rpm for 4 hours. The cells were then harvested and frozen for later use. The OD ₆₀₀ at the time of harvest was approximately 5.5-8.0.

Resuspend the cells in a ratio of 1 kg cell paste/4 L of 50 mM Tris pH 8, 100 mM NaCl buffer with or without 15-25 U/ml DNAse. Mix the cell suspension overnight at 4°C (50-60 rpm). Pass the cell suspension through a pre-cooled homogenizer on ice three times. Check the efficiency of cell lysis under a microscope, and use the unbroken cell suspension as a control. Centrifuge the cells at 4,000 rpm for 40 min in a 1 L bottle at 4°C. Save the supernatant and pellet for SDS-PAGE characterization. Analyze the expression of OXDC by SDS-PAGE. Most OXDC are found in the pellet, including inclusion bodies and other precipitates. Harvest the pellet by centrifugation at 4000 rpm for 40 min and use it immediately or freeze it at -70°C for later use.

From an 800 L fermentation reaction, we obtained 5,000 to 5,500 g of cells, which produced 2,800 to 3,000 g of wet weight pellets.

Example 2. Crystallization of oxalic acid from solubilized particles using mild denaturant concentration and subsequent anion exchange chromatography Decarboxylase.

OXDC particles stored at -20°C were used to prepare OXDC crystals.

In this procedure, particles are solubilized under mild conditions of denaturant concentration and pH. The solubilized protein is then refolded using an anion exchange matrix column.

The particles were dissolved in 2M urea, 100mM Tris pH 10.0, 10mM DTT, and 100mM NaCl (1:10 w/v). The solution was stirred at room temperature (RT) for 2 hours, then centrifuged at 15K for 30 minutes at 4°C. The supernatant was carefully decanted and stored. The particles were carefully weighed and stored separately.

Under constant and gentle stirring, the solubilized particles in the supernatant were added dropwise to 10 volumes of a solution consisting of 2M urea, 100mM Tris pH 8.0, 1mM DTT, and 1mM MnCl ₂ at a flow rate of 10ml/min. After the addition of the supernatant was complete, the protein solution was incubated at room temperature for 1 hour. After incubation, the protein solution was prepared for anion exchange chromatography by centrifugation at 15K for 30 minutes at 4°C to remove any possible precipitate.

An anion exchange chromatography column was prepared by packing a Q agarose matrix in a glass column. The column was connected to an FPLC and balanced by washing with 10 column volumes (CV) of 0.5M urea, 100mM Tris pH 8.0, 1mM DTT, and 1mM MnCl ₂ solution. The flow velocity was maintained at 6ml/min. The dissolved particles were loaded onto the column. The protein load was 8-10mg/ml matrix. After loading the sample, the column was washed with at least 10 column volumes of 100mM Tris pH 8.0, 1mM DTT, and 1mM MnCl ₂ at a flow velocity of 6ml/min. This step removed urea from the bound protein sample and allowed the protein to refold into its native conformation.

The protein was eluted from the column by discontinuous gradient or step elution with 1 M NaCl, 100 mM Tris pH 8.0, and 1 mM DTT. The protein elution was monitored at 280 nm and 10 ml fractions were collected. Peak fractions were combined and tested by SDS-PAGE and enzyme activity.

The peak fraction containing OXDC was concentrated to 15 mg/ml by agitating the cells using a 10,000 MWCO membrane and dialyzing against 10 volumes of 100 mM Tris pH 8.0, 100 mM NaCl, and 1 mM DTT. The buffer was changed twice at 1-hour intervals, and after the third buffer change, dialysis was continued overnight to maximize crystal recovery. The crystallized protein was recovered in the dialysis bag by centrifuging the sample at 2K for 15 minutes at 4°C. After dialysis, approximately 70% of the refolded OXDC crystallized. The crystals were cubic in shape and of uniform size. The crystals exhibited approximately 44 units of activity.

Example 3. Decarboxylation of oxalic acid by dissolving particles using high denaturant concentration and subsequent anion exchange chromatography Enzyme crystallization.

Using this method, the particles were dissolved in 5M urea, 50mM Tris pH 8.6, 100mM NaCl, and 10mM DTT (1:5 w/v). The solution was stirred at room temperature for 2 hours, then centrifuged at 4°C and 15K for 30 minutes. The supernatant was carefully decanted and stored. The particles were carefully weighed and stored separately.

An anion exchange chromatography column was prepared by packing a Q-Sepharose matrix into a glass column. The column was connected to an FPLC and equilibrated by washing with 3 column volumes (CV) of 4 M urea, 100 mM Tris pH 8.6, and 10 mM DTT. The column was further washed with 7 column volumes of 100 mM NaCl, 50 mM Tris pH 8, 1 mM MnCl ₂ , 10 mM DTT and eluted in a single step with 3 column volumes of 0.5 M NaCl, 50 mM Tris pH 8.0, 1 mM DTT, and 1 mM MnCl ₂ .

Appropriate fractions were collected and the protein was identified by SDS-PAGE and activity assay. Soluble protein was concentrated in the presence of high salt (0.5M NaCl). A 450ml total volume was eluted with 0.5M salt, filtered and concentrated to 45ml through pellicon, and then introduced into 100mM NaCl, 50mM Tris pH 8.0, and 1mM DTT. After protein concentration, the protein was diluted to reduce the salt concentration from 0.5M to 0.1M NaCl. At this point, OXDC crystals began to form. In this embodiment, the volume was adjusted to 210mL in 100mM NaCl, 50mM Tris pH 8.0, and 1mM DTT. The crystals formed were rotated and recovered in 100mM NaCl and 50mM Tris pH 8.0 with or without 1mM DTT.

Example 4. Use of high pH and mild denaturant concentration followed by hollow fiber concentration to dissolve particles Oxalate decarboxylase crystals.

The pellet (3.93 kg) containing inclusion bodies and other precipitates was dissolved in 9.5 L of 50 mM Tris pH 12, 500 mM NaCl, 2 M urea, and 10 mM DTT at room temperature for 2 hours. The final volume was 12 L, pH 9.9. The sample was spun at 7000 rpm for 45 minutes, and the supernatant was recovered. The total volume after spinning was 11.1 L, pH 9.9. The sample was concentrated to 5 L on a hollow fiber for 1 hour, then the volume was slowly adjusted to 20 L with 50 mM Tris pH 8.0, 500 mM NaCl, 2 M urea, and 10 mM DTT. This process was continued for 1 hour. At this point, the final urea concentration was estimated to be approximately 0.5 M urea. The volume was concentrated to 6 L on the hollow fiber for 2.5 hours. The sample was then diluted to 24 L with 50 mM Tris pH 8.0, 500 mM NaCl, 1 mM DTT, ₁ mM MnCl₂, and 200 mM L-arginine for 30 minutes. At this point, the urea concentration was estimated to be 125 mM. Another round of concentration was performed to a final volume of 5 L. The sample was diluted to 18 L with 50 mM Tris, pH 8.0, 500 mM NaCl, ₁ mM DTT, and 1 mM MnCl₂ and concentrated again to 6.5 L. The pH was now 8.1. The sample was spun at 7000 rpm for 45 minutes, and the final pellet was saved for analysis. After centrifugation, a dilution step was performed with 50 mM Tris, pH 8.0, 1 mM DTT to obtain crystals. At room temperature, with mixing, the sample was diluted to 30 L using a peristaltic pump. The flow rate of the dilution was estimated to be 50 ml/min. This dilution was performed over approximately 9 hours. The crystals were collected by centrifugation, and the supernatant was saved for analysis. The crystals were washed three times with 50 mM Tris, 100 mM NaCl, pH 8, and resuspended in 50 mM Tris, 100 mM NaCl, pH 8. The crystals were stored at 4°C.

Example 5. Crystallization of oxalate decarboxylase from cell extracts using high salt

(1) Use high salt concentration to dissolve the protein-containing particles, then concentrate and dilute them for crystallization

At room temperature, the frozen pellets (465 g) from Example 1 were dissolved in 2.3 L of 100 mM Tris, 1 mM L-cysteine HCl, 0.5 M NaCl, pH 8.0 for 2 hours to form soluble oxalate decarboxylase. The sample was spun at 7000 rpm for 45 minutes, and the supernatant was recovered. The final volume was 2.15 L, and the measured protein concentration was 24.14 mg/ml. The sample was concentrated to 550 ml by tangential flow filtration (10 kD PaI) for 1 hour, then diluted to 2,750 L with 100 mM Tris, pH 8.0, 1 mM L-cysteine HCl over 30 minutes at a flow rate of 73 ml/min and stirred at room temperature for 1 hour. Crystals formed overnight in a cold room. The crystals were harvested by centrifugation, and the supernatant was preserved for analysis. The crystals were washed three times with 100 mM Tris, 100 mM NaCl, pH 8, and then resuspended in 100 mM Tris, 100 mM NaCl, pH 8. Crystals were stored at 4° C. Recombinant Bacillus subtilis OXDC purified from E. coli expression culture medium exhibited a specific activity of approximately 50-60 U/mg under standard assay conditions (see Example 15).

(2) Crystallization by dissolving the particles using high salt concentration, followed by concentration and dialysis

At room temperature, the frozen particles (510g) of Example 1 were dissolved in 3L 100mM Tris, 1mM L-cysteine HCl, 0.5M NaCl, pH 8.0 for 2 hours. The sample was rotated at 7000rpm for 30 minutes and the supernatant was recovered. The sample was concentrated to 500ml for 1 hour by tangential flow filtration (10kDPall) and then dialyzed in 100mM Tris pH 8.0 under stirring. Crystals were formed overnight in a cold room. The crystals were harvested by centrifugation and the supernatant was preserved for analysis. The crystals were washed 3 times with 100mM Tris, pH 8.0 and then resuspended in 100mM Tris pH 8.0. The crystals were preserved at 4°C. The crystal yield was 60%.

(3) Crystallization by dissolving the particles using high salt concentration, followed by concentration and dialysis

At room temperature, the frozen pellets (510 g) of Example 1 were dissolved in 3L 100 mM Tris, 1 mM L-cysteine HCl, 0.5 M NaCl, pH 8.0 for 2 hours. The sample was rotated at 7000 rpm for 30 minutes and the supernatant was recovered. The sample was concentrated to 500 ml for 1 hour by tangential flow filtration (10 kDPall) and then dialyzed in 100 mM Tris pH 7.5 under stirring. Crystals were formed overnight in a cold room. The crystals were harvested by centrifugation and the supernatant was preserved for analysis. The crystals were washed 3 times with 100 mM Tris, pH 7.5 and then resuspended in 100 mM Tris pH 7.5. The crystals were stored at 4°C. The crystal yield was 67%.

(4) Crystallization by dissolving the particles using high salt concentration, followed by concentration and dialysis

At room temperature, the frozen pellets (510 g) of Example 1 were dissolved in 3 L 100 mM Tris, 1 mM L-cysteine HCl, 0.5 M NaCl, pH 8.0 for 2 hours. The sample was rotated at 7000 rpm for 30 minutes and the supernatant was recovered. The sample was concentrated to 500 ml for 1 hour by tangential flow filtration (10 kDPall) and then dialyzed in 100 mM Tris pH 7.0 under stirring. Crystals were formed overnight in a cold room. The crystals were harvested by centrifugation and the supernatant was preserved for analysis. The crystals were washed 3 times with 100 mM Tris, pH 7.0 and then resuspended in 100 mM Tris pH 7.0. The crystals were stored at 4°C. The crystal yield was approximately 80%.

(5) Crystallization by dissolving the particles using high salt concentration, followed by concentration and dialysis

At room temperature, the frozen particles (510g) of Example 1 were dissolved in 3L 100mM Tris, 1mM L-cysteine HCl, 0.5M NaCl, pH 8.0 for 2 hours. The sample was rotated at 7000rpm for 30 minutes and the supernatant was recovered. By tangential flow filtration (10kDPall), the sample was concentrated to 500ml for 1 hour and then dialyzed in 100mM sodium citrate buffer pH 6.5 under stirring. Crystals were formed overnight in a cold room. The crystals were harvested by centrifugation and the supernatant was preserved for analysis. The crystals were washed 3 times with 100mM sodium citrate buffer pH 6.5 and then resuspended in 100mM sodium citrate buffer pH 6.5. The crystals were preserved at 4°C. The crystal yield was about 70%.

(6) Crystallization by dissolving the particles using high salt concentration, followed by concentration and dialysis

At room temperature, the frozen particles (510g) of Example 1 were dissolved in 3L 100mM Tris, 1mM L-cysteine HCL, 0.5M NaCl, pH 8.0 for 2 hours. The sample was rotated at 7000rpm for 30 minutes and the supernatant was recovered. By tangential flow filtration (10kDPall), the sample was concentrated to 500ml for 1 hour and then dialyzed in 100mM sodium citrate buffer pH 6.0 under stirring. Crystals were formed overnight in a cold room. The crystals were harvested by centrifugation and the supernatant was preserved for analysis. The crystals were washed 3 times with 100mM sodium citrate buffer pH 6.0 and then resuspended in 100mM sodium citrate buffer pH 6.0. The crystals were preserved at 4°C. The crystal yield was about 60%.

(7) After homogenization and dissolution, OXDC from cell paste was crystallized

The cells were resuspended at a ratio of 1 kg cell paste/3 L buffer containing 100 mM Tris pH 7.5, 500 mM NaCl, 5 mM cysteine, and 1 mM manganese chloride. The cell suspension was mixed (50-60 rpm) overnight at 4°C. The cell suspension was passed through a pre-cooled homogenizer on ice twice. The efficiency of cell lysis was checked under a microscope, and the unbroken cell suspension was used as a control. The suspension was made up to 10 L and mixed with an overhead stirrer at room temperature for 3 hours. The crude extract was then centrifuged at 7,000 rpm for 30 min at 4°C, and the supernatant was recovered. The supernatant and pellet were saved for SDS-PAGE characterization. The expression of OXDC was analyzed by SDS-PAGE. Most OXDC was found in the supernatant. The final volume was 10 L, and the measured protein concentration was 34 mg/ml. The sample was concentrated to 3.5 L by tangential flow filtration (10 kD Pall) for 1 hour and then diluted with crystallization buffer (100 mM Tris, 100 mM NaCl pH 7.5) at room temperature under stirring for 1 hour. The crystals were harvested by centrifugation and the supernatant was saved for analysis. The crystals were washed 3 times with 100 mM Tris, 100 mM NaCl pH 7.5 and then resuspended in 100 mM Tris and 100 mM NaCl, pH 7.5. The crystals were stored at 4°C.

Example 6. Crystallization of OXDC from soluble protein .

OXDC was expressed in Escherichia coli in shake flasks. Cells were lysed using a microfluidizer using 25 mM Tris-HCl buffer, pH 8.0, and 100 mM NaCl containing 25 U/mL DNase I. The cell lysate was incubated at room temperature for 1 hour to allow OXDC crystals to form. The crystals were spun and reconstituted in 100 mM Tris, 100 mM NaCl, pH 8.

Example 7. Crystallization of OXDC by vapor diffusion.

Hanging drop crystallization experiments were performed using commercially available sparse matrix crystallization kits: Crystal Screen (Hampton Research; Aliso Viejo, CA), Crystal Screen 2 (Hampton Research), Wizard I (Emerald Biosystems; Bainbridge Island, WA), Wizard II (Emerald Biosystems), Cryo I (Emerald Biosystems), and Cryo II (Emerald Biosystems).

600 μl of reagent was placed in each well. 3 μl of reagent was dropped onto a microscope glass coverslip, and 3 μl of OXDC was dropped into the reagent droplet and gently mixed. From this 6 μl reagent and OXDC droplet, another 5 droplets were prepared. As the droplets were gently mixed, each subsequent (smaller) droplet had a different and unknown protein/reagent ratio, thereby increasing the possibility of obtaining crystals in a short period of time. After incubation at room temperature overnight, the crystals of the hanging drop were examined under a microscope. A large number of crystallization conditions were obtained, as shown in Table 1.

Table 1: Crystallization conditions of OXDC in hanging drops ^.

aOXDC concentration was approximately 1.7 mg/mL ^as determined by Bradford assay.

Example 8. Crystallization of OXDC by microbatch.

Oxalate decarboxylase was crystallized from a number of crystallization conditions via a microbatch approach:

(i) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 10 μl of 16% PEG 8000. Crystallization occurred immediately within 2-5 seconds. Large crystals formed with some precipitate.

(ii) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 10 μl of 20% PEG 8000. Crystallization occurred immediately within 2-5 seconds. Large crystals formed without precipitate.

(iii) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 10 μl of 24% PEG 8000. Crystallization occurred within 2-5 seconds, forming smaller, cubic crystals.

(iv) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 10 μl of 28% PEG 8000. Crystallization occurred immediately within 2-5 seconds. Very small, cubic crystals were formed. No precipitation was observed.

(v) 8 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 12 μl of 24% PEG 8000. Crystallization occurred immediately within 2-5 seconds. Very small, cubic crystals were formed. No precipitation was observed.

(vi) 9 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 11 μl of 24% PEG 8000. Crystallization occurred immediately within 2-5 seconds. Small cubic crystals were formed. No precipitation was observed.

(vii) 10 μl of purified oxalate decarboxylase at a concentration of 23.46 mg/ml was mixed with 10 μl of 24% PEG 8000. Crystallization occurred within 2-5 seconds. Small cubic crystals were formed. No precipitation was observed.

(viii) 11 μl of purified oxalate decarboxylase at a concentration of 23.46 mg/ml was mixed with 9 μl of 24% PEG 8000. Crystallization occurred within 2-5 seconds. Cubic crystals were formed. No precipitation was observed.

(ix) 12 μl of purified OXDC at a concentration of 23.46 mg/ml was mixed with 8 μl of 24% PEG8000. Crystallization occurred immediately within 2-5 seconds. Cubic crystals were formed. No precipitation was observed.

Example 9. Activity of Soluble OXDC and OXDC Crystals

After dissolving the pellet, soluble OXDC was collected, spun, and the supernatant recovered as described in Example 5. After harvesting and washing the crystals, OXDC crystals were collected as described in Example 5. The activity of soluble OXDC and OXDC crystals was measured according to Example 15. In one experiment, the activity of soluble OXDC was 12 units/mg and the activity of OXDC crystals was 35 units/mg.

Soluble OXDC is collected as described in any of Examples 2-5, and OXDC crystals are harvested as described in any of Examples 2-8. The activity of the soluble and crystalline OXDC is measured according to Example 15. The activity of the OXDC crystals can be at least about 100%, 200%, 300%, 400%, or 500% of the activity of the soluble OXDC.

Example 10. Cross-linking of oxalate decarboxylase crystals using glutaraldehyde.

Oxalate decarboxylase crystals prepared according to any of Examples 2-8 were cross-linked using glutaraldehyde. After crystallization, the OXDC crystals were concentrated to 20-30 mg/ml. A 1% glutaraldehyde solution was prepared by adding 0.8 ml of 25% glutaraldehyde to 20 ml of crystals. The crystals were tumbled at room temperature for 18 hours. The cross-linked crystals were washed five times with 100 mM Tris, pH 7.00, and resuspended in 10 mM Tris, pH 7.00.

Comparison of the specific activities of crystalline OXDC and cross-linked OXDC (referred to as OXDC-CLEC) (6 experiments) showed that in different preparations, the cross-linked oxalate decarboxylase crystals retained greater than 30% to greater than 50% of the original activity of the crystalline protein.

To test the effect of different concentrations of glutaraldehyde on enzyme activity, 1 ml aliquots of OXDC crystals (60 mg/ml) were cross-linked with different concentrations of glutaraldehyde (from 0.05% to 2% final concentration) at pH 8.0 at 25° C. for 18 hours. The cross-linked crystals were isolated by centrifugation at 2000 rpm in an Eppendorf tube to terminate the cross-linking and then resuspended in 1 ml of 100 mM Tris·HCl, pH 7.0. The cross-linked OXDC (OXDC-CLEC) was then washed five times with 100 mM Tris·HCl buffer, pH 7.5, and then three times with 10 mM Tris·HCl buffer, pH 7.5 (see results in Table 2 below).

Example 11. pH controlled solubility of cross-linked oxalate decarboxylase crystals.

The solubility of various cross-linked oxalate decarboxylase crystals was examined by lowering the pH from 7.5 to 3.0. The cross-linked crystals were incubated at 1 mg/ml in 50 mM glycine HCl (pH 3.0). After incubation at 37°C for 5 hours with stirring, an aliquot was removed. Undissolved cross-linked crystals were separated by centrifugation at 2000 rpm and the supernatant was filtered through a 0.22 μm filter. The soluble protein concentration was measured at OD ₂₈₀ nm. The results are summarized in Table 2 below.

Table 2: pH-controlled solubility of OXDC crystals and OXDC-CLEC cross-linked with different percentages of glutaraldehyde

sample % glutaraldehyde % protein extraction OXDc-CLEC-1 0.005 100.0 OXDC-CLEC-2 0.010 100.0 OXDC-CLEC-3 0.050 2.2 OXDC-CLEC-4 0.075 0.0 OXDC-CLEC-5 0.100 0.0 OXDC-CLEC-6 0.200 0.0 OXDC-CLEC-7 1.000 0.0

These results indicate that substantially stable glutaraldehyde OXDC cross-linked crystals are formed in the presence of at least about 0.05% (final concentration) glutaraldehyde.

Example 12. pH Activity Characteristics of Soluble OXDC, Crystalline OXDC, and OXDC-CLEC

Oxalate decarboxylase crystals prepared as described in Example 2-8 were cross-linked by the addition of glutaraldehyde (Sigma). A 1 ml aliquot of OXDC crystals (30-40 mg/ml) was cross-linked with 1% glutaraldehyde (final concentration) at pH 8.0 at 25°C for 18 hours. The cross-linked crystals were isolated by centrifugation at 2000 rpm in an Eppendorf tube to terminate cross-linking and then resuspended in 1 ml of 100 mM Tris-HCl, pH 7.0. OXDC-CLEC was then washed five times with 100 mM Tris-HCl buffer, pH 7.0, followed by three washes with 10 mM Tris-HCl buffer, pH 7.0. The pH activity profile of OXDC-CLEC was determined by measuring the activity of the crystals as described in Example 15 using different buffers and pH values: 50 mM glycine·HCl buffer at pH 2.0 and 3.0; 50 mM succinate buffer at pH 4.0, 5.0, and 6.0; and 50 mM Tris buffer at pH 7.0. The activity level was measured twice at each pH, and the average activity was calculated. The results, shown in Figure 1, demonstrate that OXDC-CLEC is more active than its soluble counterpart between pH 3.5 and 6.0. Uncross-linked crystals exhibited significantly higher activity than the soluble form of oxalate decarboxylase at various pH values, ranging from about 50% to about 200%, 300%, or 400% higher.

Example 13. Oxalate decarboxylase treatment in an animal model of enterogenic hyperoxaluria.

Male Sprague Dawley (SD) rats fed a high- oxalate diet constitute an animal system suitable for studying enterogenic hyperoxaluria. In this study, administration of 1.1% dietary potassium oxalate resulted in a 5- to 10-fold increase in urinary oxalate.

Twenty Sprague Dawley (SD) rats, less than 35 days old and weighing 100-120 grams, were randomly divided into a control group and an experimental group (5 rats per group). Prior to treatment, the rats were acclimated to their respective metabolic cages (Lab Products, Inc.; Seaford, DE) for 7 days. During this period, the rats were given ad libitum access to supplemented acidified water and fed a synthetic diet containing 1.1% potassium oxalate and a low (0.5%) concentration of calcium (Research Diets TD89222PWD; Harlan Teklad; Madison, WI). The rats maintained this diet during the treatment period.

After the acclimation phase, three different doses of recombinant oxalate decarboxylase formulated as cross-linked crystals with 1% glutaraldehyde (see, e.g., Example 9) were administered to experimental rats for four consecutive weeks. The crystals were orally administered as a solidified/dried food enzyme mixture (5, 25, and 80 mg OXDC-CLEC slurry in 10 mM Tris·HCl pH 7.0, each independently mixed with 15 g of food and solidified/dried; each morning, the food container was refilled with approximately 20 g of food/enzyme mixture). Prior to treatment, rats were randomly divided into control and experimental groups based on their basal urine oxalate.

Analysis of urine samples: 24-hour urine samples were collected in metabolic cages on acid (250 μl of 6N hydrochloric acid was mixed with the urine samples collected over 24 hours) to minimize spontaneous decomposition of urinary ascorbic acid to oxalate. Samples were stored at -70°C before further analysis. Daily urine and multiple 24-hour urine samples were collected for oxalate and creatinine measurements. Oxalate and creatinine were determined as described in Example 15. Urinary excretion of oxalate and creatinine was expressed as μmol of oxalate and creatinine detected in the 24-hour urine sample. All data were statistically analyzed using the Student's t-test.

As shown in Figure 2, oral administration of OXDC-CLEC to SD rats with chronic hyperoxaluria resulted in a sustained decrease in urinary oxalate starting on day 4 of treatment. A maximum continuous decrease of 25-40% was recorded in the highest dose group (80 mg OXDC-CLEC). Lower doses of 5 mg and 25 mg OXDC-CLEC resulted in smaller decreases in urinary oxalate (up to 30% in the 25 mg group and up to 20% in the 5 mg group). The 25 mg and 80 mg doses produced significant decreases on all days tested (except day 21 in the 25 mg group), while the lowest dose of 5 mg had a minimal, insignificant effect. These results reveal a dose-dependent effect of OXDC-CLEC treatment.

Example 14: Oral oxalate decarboxylase treatment in an animal model of primary hyperoxaluria.

Mouse Model of Type I Primary Hyperoxaluria: AGT1 knockout mice lack the hepatic peroxidase alanine:glyoxylate aminotransferase, a defect that causes type I primary hyperoxaluria. Chimeric mice were bred to homozygous lines in the C57Bl6 and 129/sv background strains. All homozygous Agxt mice exhibited mild hyperoxaluria (1-2 mmol/L), with urine oxalate elevated 5-10 times above normal compared to wild-type (0.2 mmol/L). It was also found that 30-50% of males and 0% of females developed mild nephrocalcinosis and urinary calcium oxalate stones later in life (4-7 months of age). Interestingly, when the mutation was analyzed in the same C57Bl6 strain, hyperoxaluria in either sex was not associated with the development of urinary stones; this emphasizes the phenotypic variability often observed in this disease.

A total of 44 male mice (AGT1 KO/129sv strain, developed by Dr. Salido, La Laguna, Tenerife, Spain) were used in these experiments. The mice were randomly divided into a control group and three experimental groups. The mice weighed 20-25 grams and were less than 6 months old.

AGT1 KO (129sv) mice were challenged with ethylene glycol (EG) to induce severe hyperoxaluria and the formation of calcium oxalate deposits in the renal parenchyma. EG is a common alcohol metabolized to oxalate in the liver. Typically, 2-6 weeks after EG challenge, AGT1 KO mice in the 129/sv background exhibit signs of impaired renal function, as measured by: (i) altered excretion of oxalate in the urine; (ii) decreased creatinine clearance, and (iii) nephrocalcinosis, which ultimately leads to renal failure and death.

Prior to treatment, mice were acclimated to individual metabolic cages (Tecniplast USA Inc, Exton, PA, USA) for 7 days and fed a standard breeder diet (17% protein, 11% fat, 53.5% carbohydrates) containing less than 0.02-0.08% oxalate and approximately 0.5-0.9% calcium. After the acclimation period, the mice were divided into four groups; three treatment groups were fed oxalate decarboxylase-CLEC mixed with food, while mice in a matched control group received the same diet without the experimental substance. From the first day of treatment until the end of the study, all mice were given drinking water supplemented with 0.7% EG ad libitum. Several days after the challenge, the mice excreted approximately 3-6 mmol/L oxalate in their urine daily, which was approximately 10-20 times higher than that of wild-type (unchallenged) mice.

Administration of OXDC-CLEC Enzyme; Dose-Ranging Study: In the OXDC-CLEC dose study, a total of 44 male mice from the AGT1KO/129sv strain were used. Mice weighed 20-25 grams and were less than 6 months old. The mice were challenged with EG and then randomly divided into a control group and an experimental group. The efficacy of three different doses of recombinant oxalate decarboxylase formulated as cross-linked crystals (1% glutaraldehyde; see Example 9) was monitored for four consecutive weeks. The term "OXDC-CLEC" used in this example refers to recombinant oxalate decarboxylase formulated as cross-linked crystals (1% glutaraldehyde) as described in Example 9. OXDC-CLEC was orally administered as a coagulated/dried food enzyme mixture at nominal doses of 5, 25, and 80 mg/day. A sufficient amount of enzyme slurry in 10 mM Tris-HCl buffer (pH 7.0) was mixed with 3.5 g of food and coagulated/dried. Each morning, the food container was refilled with approximately 7 g of the food/enzyme mixture.

Evaluation of the efficacy of OXDC-CLEC : The efficacy of enzyme treatment was monitored by reduction of urinary oxalate, prevention of calcium oxalate deposition in the renal parenchyma, and survival. At the end of the study, surviving mice were sacrificed and blood samples were taken for creatinine measurement.

Analysis of urine samples: 24-hour urine samples were collected in metabolic cages over acid (50 μl 6N hydrochloric acid/3-4 ml urine) to minimize spontaneous decomposition of urinary ascorbic acid to oxalate. Urine samples were stored at -20°C until further analysis. Daily urine and multiple 24-hour urine samples were collected and analyzed for oxalate and creatinine levels. Oxalate and creatinine were determined as described in Example 15. Urinary excretion of oxalate and creatinine was expressed as μmol of oxalate and creatinine excreted in the 24-hour urine sample (mL). Data were statistically analyzed using the Student's t-test.

Analysis of blood samples : At the end of the study, mice were sacrificed and serum samples were collected. For serum creatinine measurement, a slightly modified Jaffe reaction method was used (see, for example, creatinine microassay plate kit from Oxford Medical Research, Inc.; Slot, Scand J. Clin. Lab. Invest. 17 : 381, 1965; and Heinegard D, Clin. Chim. Acta 43: 305, 1973). 80 μl of undiluted serum sample was mixed with 800 μl of picrate in a test tube and incubated at room temperature for 30 minutes. Color development was measured spectrophotometrically at 510 nm; 33.3 μl of 60% acetic acid was then added to quench nonspecific reactions. The sample was thoroughly mixed and, after incubation at room temperature for 5 minutes, read again at 510 nm. The final absorbance was expressed as the difference between the two readings. Serial dilutions of 1 mM creatinine solution were used for the standard curve.

Renal function is indirectly measured by measuring creatinine clearance. Creatinine clearance is expressed as the rate of creatinine excretion (U _cr x V) (where U _cr represents the creatinine concentration in the urine sample (μmol/L)) divided by plasma creatinine (P _cr ). This is expressed as:

C _cr =(U _cr xV)/P _cr =mL/h

Safety parameters monitored during the study were mortality, food and water intake, and body weight. Mortality checks and cage-side objective observations were performed daily during the study. Food intake was measured daily, and water intake was recorded weekly. Body weights were recorded for all animals at the beginning and end of the study.

As shown in Figure 3, oral administration of OXDC-CLEC to EG-challenged AGT1KO (129sv) mice resulted in a significant reduction in urinary oxalate levels from day 4 of treatment to the end of the study compared to matched untreated control mice. Reductions ranging from 30 to 50% were observed in all three treatment groups, with the greatest reduction observed in the highest dose group (80 mg OXDC-CLEC). Lower doses of 25 mg and 5 mg OXDC-CLEC produced reductions in urinary oxalate of up to 35%.

Results were analyzed using an unpaired two-tailed Student's t-test. At the start of the study, each dosing group had n=11 mice, but several mice died from ethylene glycol attacks during the study. The results shown only include mice that survived the specific day of urine oxalate measurement. Observing that the initial rise in urine oxalate leads to nephrocalcinosis, reduced renal filtering function, and subsequent oxalate excretion rate reduction over time and, in the worst case, final death can best explain the bell-shaped curve of urine oxalate excretion in the control group.

Renal function was assessed by creatinine clearance measurement. At the end of the study, all animals that survived the 4-week EG challenge were sacrificed, and blood was collected for measurement of plasma creatinine and creatinine clearance. All 11 mice in the 80 mg dose group survived the 4-week EG challenge; 8 of 11 mice in the 25 mg OXCD-CLEC dose group survived; 8 of 11 mice in the 5 mg OXCD-CLEC dose group survived; and 7 of 11 mice in the control group survived. Serum creatinine was measured using the Jaffe reaction method described above with slight modifications (see, e.g., Creatinine Microassay Plate Kit from Oxford Medical Research, Inc.; Slot, Scand J. Clin. Lab. Invest. 17 :381, 1965; and Heinegard D, Clin. Chim. Acta 43:305, 1973).

The efficacy of oral OXDC-CLEC on renal function was assessed by measuring creatinine clearance. Creatinine clearance in mice that survived the full month study period is shown in Figure 4. When compared to the control group, creatinine clearance was significantly higher in surviving mice that received 80 mg of OXDC-CLEC (p < 0.05).

All mice (11/11) in the 80 mg OXDC-CLEC treatment group survived the 4-week EG challenge regimen, while only 7 mice (7/11) in the control group survived the regimen. Renal filtration rate, as measured by creatinine clearance, was also significantly lower in the 80 mg dose group (Figure 4).

Renal tissue pathological analysis: paraffin embedding is routinely processed mouse kidney, and locates, so as to obtain the complete cross section of kidney.With each slice 4 μm, each kidney is cut into 12 serial sections, stained with hematoxylin and eosin, for conventional histological examination, or by specific Yasue metal replacement histochemical method to detect the presence of calcium oxalate crystals in kidney tissue.Using 20X magnification, microscope slides are examined under a microscope, and inspectors score sections under 4-class scales, and the same standard is applied to each anatomical region (cortex, medulla and papilla) of kidney.Scoring is (i) without (no oxalate crystals in any region)；(ii) minimum (1-5 crystals are present in any region)；(iii) medium (6-10 crystals are present in any region)；and (iv) serious (all regions have multiple crystal collections).

Representative images of renal tissue from treated and control animals are shown in Figures 5A-5C. At 20x magnification, Yasue-positive calcium oxalate crystals were visible in the renal parenchyma. All mice in the treatment group treated with 80 mg of OXDC-CLEC had normal, healthy kidneys with no trace of calcium oxalate deposits (Figure 5A). Moderate nephrocalcinosis (Figure 5B) and severe nephrocalcinosis (Figure 5C) were observed in the control group and in some mice from the low-dose treatment group. The white arrows in Figure 5C indicate calcium oxalate deposits, and the gray arrows indicate large areas of interstitial fibrosis.

Histological examination of the kidneys using the specific Yasue metal replacement method revealed that deposits were primarily present in the cortex and medulla of the kidneys. In cases of severe nephrocalcinosis (Figure 5C), calcium oxalate deposits were randomly distributed in the kidneys. Signs of fibrosis and inflammation were also observed, with altered glomerular morphology and occasional formation of calcium oxalate deposits in the glomeruli. Following necropsy, all mice (11/11) from the 80 mg dose group had normal kidney morphology, with no trace of calcium oxalate deposits in the kidneys or bladder. In contrast, 100% (11/11) of the mice from the untreated control group had calcium oxalate deposits. In the low-treatment groups (25 mg or 5 mg OXDC-CLEC), 63% (7/11) of the mice had calcium oxalate deposits in the kidneys. These results confirm the positive, dose-dependent effect of oral OXDC-CLEC treatment on the reduction of hyperoxaluria and the prevention of calcium oxalate crystal deposition in the kidneys of EG-challenged AGT1 KO mice. A summary of the histopathological analysis of mice from all four groups is shown in Table 3 .

Table 3. Severity of nephrocalcinosis and the number of affected mice in the treated and control groups after oral treatment with OXDC-CLEC. number

The efficacy of oral OXDC-CLEC treatment on the frequency of urinary stone formation in control mice and mice from three different treatment groups was also evaluated. Two major types of calcium were found in the bladders of EGAGT1KO mice: calcium oxalate monohydrate stones and calcium oxalate dihydrate stones. Visible bladder stones were present in 36% (4/11) of mice in the control group and in 19% (2/11) of mice in the two lower treatment groups. No bladder stones were observed in the high-dose OXDC-CLEC group (80 mg). X-ray diffraction analysis showed that stones with a whitish, rough, bud-like surface were primarily composed of calcium oxalate monohydrate, while relatively larger stones with sharp crystal corners corresponded to calcium oxalate dihydrate.

Survival analysis was performed using the Kaplan-Meier estimator. The effect of OXDC-CLEC treatment on the survival of ethylene glycol-challenged mice was analyzed using the Kaplan-Meier method, in which the survival rate of subjects who died at a certain time point was divided by the number of subjects still alive at that time point in the study. This method graphically explains the differences between the groups in the study (Figure 6). Statistical programs such as Kaleida Graph and STATS are often used for calculations.

Oral treatment with OXDC-CLEC increased the survival of EG-challenged AGT1KO mice compared to matched controls. All mice (11/11) from the 80 mg OXDC-CLEC-treated group survived the 30-day study period without signs of illness, whereas 4/11 mice from the control group had severe nephrocalcinosis and developed urinary stones.

Since OXDC-CLEC is intended for oral administration, the potential for adverse effects of cross-linked crystals on gastrointestinal (GI) tissues was evaluated. The digestive tract of treated EG-challenged AGT1 KO mice was examined macroscopically, and histological analysis of different sections of the GI tract, including the stomach (primary and antral) and small intestine (jejunum and ileum), was performed using hematoxylin-eosin staining. This evaluation confirmed that oral treatment with OXDC-CLEC for 4 weeks was well tolerated and did not cause structural or morphological changes in the GI tract. Similar results were observed for the large intestine.

Summary of a dose-ranging study of EG-challenged AGT1 KO mice treated orally with oxalate decarboxylase-CLEC . Oral treatment with OXDC-CLEC is safe and effective in an animal model of primary hyperoxaluria. In summary, 4 weeks of oral treatment with OXDC-CLEC reduced urinary oxalate by 30-50%. Significant and sustained reductions were documented across the three doses evaluated. 4 weeks of oral treatment at the highest therapeutic dose prevented calcium oxalate deposition in the renal parenchyma. 4 weeks of oral treatment at two lower doses improved survival and prevented animal mortality at the highest dose studied. Finally, the 4-week treatment regimen produced no macroscopic or microscopic changes in the gastrointestinal tract.

Example 15. Determination

Protein concentration determination: The concentration of oxalate decarboxylase was determined by measuring absorbance at 280 nm. An absorbance of 1.36 optical density (OD) was considered to be 1 mg/ml.

OXDC Activity Assay: The activity of soluble oxalate decarboxylase, oxalate decarboxylase crystals, and cross-linked oxalate decarboxylase crystals (OXDC-CLEC) was measured using a modified Sigma-Aldrich protocol (enzyme assay for oxalate decarboxylase EC 4.1.1.2). This is an indirect, two-step activity assay; in the first reaction, OXDC converts the substrate oxalate into formate and carbon dioxide. In the second reaction, formate hydrogen is stochiometrically transferred to NAD by formate dehydrogenase, forming NADH. The subsequent concentration of NADH is quantified spectrophotometrically at 340 nm. The unit of enzyme activity (u) is defined as follows: One unit of oxalate decarboxylase forms formate and carbon dioxide from 1.0 μmol oxalate per minute at pH 5 and 37°C.

Assay samples were standardized in 5 mM potassium phosphate buffer, pH 7.0, containing 1 mM DTT (Sigma) at concentrations of 0.007-0.02 mg/mL for OXDC crystals and 0.009-0.03 mg/mL for OXDC-CLEC. Protein concentration was determined by absorbance at 280 nm. Formate dehydrogenase (FDH) was prepared in cold _diH₂O at 40 U/mL and kept on ice before use. All other reagents were kept at room temperature. Reagents were added to 2 ml microtubes using a stir bar as follows: 300 μl of 100 mM potassium phosphate and 200 μl of potassium oxalate, pH 4.0, were mixed and warmed in a water bath at 37°C for 5 min. Then, 100 μl of 5 mM potassium phosphate containing 1 mM DTT was added to a blank vial and 100 μl of diluted oxalate decarboxylase was added to all other vials. After 2 minutes, the reaction was terminated with 150 mM dipotassium phosphate. In the second reaction, 25 μl of NAD solution was added to 100 μl of FDH solution, and incubation was continued for an additional 20 min. All samples were then centrifuged at 16,100 rpm for 1 min. The reaction mixture was then transferred to a 1.5 mL UV cuvette, and the absorbance at 340 nm was measured and recorded using a Shimadzu BioSpec (Shimadzu Scientific Instruments, Columbia, Maryland).

The enzyme specific activity was calculated as follows:

In one specific experiment, cross-linked oxalate decarboxylase crystals (OXDC-CLEC) (diamond-shaped crystals cross-linked with 1% glutaraldehyde by tumbling overnight; see Example 9) were compared to OXDC crystals. OXDC-CLEC retained 50% of the activity of the corresponding crystalline OXDC preparation.

Oxalate is measured by colorimetric method: Oxalate colorimetric kits for quantitative determination of oxalate in urine are purchased from Trinity Biotech USA (St. Louis, MO) or Greiner Diagnostic AG (Dennliweg 9, Switzerland). Urine samples are diluted and processed according to the manufacturer's instructions. The determination comprises two enzymatic reactions: (a) oxalate oxidase oxidizes oxalate to carbon dioxide and hydrogen peroxide, and (b) the hydrogen peroxide thus formed reacts with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB) in the presence of peroxidase to form an indamine dye that can be detected at an absorbance of 590 nm. The intensity of the color produced is directly proportional to the concentration of oxalate in the sample. Urine oxalate values are calculated from a standard curve.

Creatinine was determined by colorimetric method: Creatinine colorimetric kits for quantitative determination of creatinine in urine were purchased from Quidel Corporation (San Diego, CA; METRA Creatinine Assay Kit) or Randox Laboratories (Antrim, United Kingdom). The assay is based on the principle that creatinine reacts with picric acid in an alkaline solution to form a product with an absorbance at 492 nm. The amount of complex formed is directly proportional to the creatinine concentration. 24-hour rat urine samples collected from individual metabolic cages were diluted 15-fold with double distilled water. 20 μl of the diluted urine sample was mixed with 20 μl of picric acid/sodium hydroxide (1:1). After incubation at room temperature for 2 minutes, the absorbance at 492 nm was measured. Urine creatinine values were calculated from a standard curve.

Example 16. OXDC Therapy for Treating Oxalate-Related Disorders in Humans

Oral administration of cross-linked oxalate decarboxylase crystals can be used to treat or prevent oxalate-related conditions, such as hyperoxaluria. Oxalate decarboxylase crystals are administered at an approximate dose of 10 μg/kg to 25 mg/kg, 1 mg/kg to 25 mg/kg, or 5 mg/kg to 100 mg/kg, as determined by the treating clinician and dependent on the severity of the symptoms and progression of the disease. The cross-linked oxalate decarboxylase crystals are administered 1, 2, 3, 4, or 5 times daily, or less frequently, such as once or twice weekly. Oral administration of OXDC-CLEC results in a reduction in urine oxalate levels of at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or more.

Example 17. Recombinant production of oxalate decarboxylase

In human embryonic kidney (HEK293) cells: DNA encoding OXDC (e.g., SEQ ID NO: 1 or 2) is cloned into a suitable expression vector. After sequence confirmation, the vector is linearized and pre-inoculated HEK293 cells are transformed with the linearized vector using Lipofectamine ^™ 2000 transfection reagent in a 6 cm diameter dish. The transfection reaction is cultured overnight in an appropriate culture medium and then transformants are selected in a culture medium supplemented with 0.5 g/L neomycin. After growing for up to 3 weeks in a culture medium containing neomycin, stably transfected HEK293 cell clones are identified. The clones are then isolated and propagated and used for OXDC expression.

In Chinese Hamster Ovary (CHO) cells: DNA encoding the OXDC gene was cloned into a suitable expression vector. The cultured CHOlec3.2.8.1 cells were then isolated by trypsinization and harvested by centrifugation. The cells were then suspended in electroporated phosphate-buffered saline (EPBS) to a final concentration of ~1x10 ⁷ /ml and transformed with the linearized vector by electroporation. After overnight incubation, the culture medium was replaced with culture medium supplemented with 0.5g/L neomycin. Continuous culture medium replacement was performed to screen for stably transfected CHO cell clones. Once stably transfected cell clones were established and propagated, the cells were used for OXDC expression.

In Pichia pastoris: DNA encoding the OXDC gene is cloned into a suitable expression vector. After sequence confirmation, the vector is linearized and then transformed into Pichia pastoris host cells (see, Whittaker et al., J. Biol. Inorg. Chem. 7 : 136-145, 2002). Transformants are selected with Zeocin, amplified in buffered glycerol complex medium (BMGY), and induced with methanol. OXDC can then be isolated from the culture medium.

In Saccharomyces cerevisiae: The synthesized OXDC gene can be cloned into a suitable expression vector containing, for example, a Gal1 promoter (pGal) and an expression terminator sequence. After sequence confirmation, the expression vector is transformed into competent Saccharomyces cerevisiae W303-1A by electroporation. Transformants are screened, propagated, and then used for OXDC expression.

In insect cells: DNA encoding OXDC can be cloned into a suitable expression vector, such as a baculovirus system. After sequence confirmation, the vector is transformed into competent DH10Bac E. coli cells. E. coli cells containing the recombinant bacmid are screened and confirmed. The recombinant bacmid DNA is isolated and used to transfect insect Sf9 cells using reagents such as Cellfectin ^™ reagent (Invitrogen, Carlsbad, California). The recombinant baculovirus particles can then be isolated, propagated, and titrated, and then used to infect Sf9 cells for OXDC expression.

In E. coli: DNA encoding OXDC can be cloned into a suitable E. coli expression vector. After sequence confirmation, the vector is transformed into competent E. coli BL21 or, if necessary, E. coli Origami B (DE3), which allows disulfide bond formation in the recombinant protein expressed in this strain. Transformants are screened by culturing on nutrient plates containing antibiotics and confirmed by colony PCR using primers specific for the OXDC gene. Transformants are then cultured in liquid culture medium and induced with isopropyl-β-D-thiogalactopyranoside (IPTG) for OXDC expression.

Example 18. Sequence

The Flammulina velutipes sequence (SEQ ID NO: 1) to be expressed in Candida boidinii . The two Not I sequences are underlined; the bold ATA triplets are spacer codons; the double-underlined sequence is the α-mating factor sequence optimized for Candida boidinii; the OXDC coding sequence is in lowercase.

1 ataagaat GCGGCCGC ATA

50

100

150

200

250 atgtttaataatt

300 ttcaaagattattaactgttattttattatctggttttatactgctggtgtt

350 ccattagcttctactactactggtactggtactgctactggtacttctac

400 tgctgctgaaccatctgctactgttccatttgcttctactgatccaaatc

450 cagttttatggaatgaaacttctgatccagctttagttaaaccagaaaga

500 aatcaattaggtgctactattcaaggtccagataatttaccaattgattt

550 acaaaatccagatttattagctccaccaactactgatcatggttttgttg

600 gtaatgctaaatggccattttctttttctaaacaaagattacaaactggt

650 ggttgggctagacaacaaaatgaagttgttttaccattagctactaattt

700 agcttgtactaatatgagattagaagctggtgctattagagaattacatt

750 ggcataaaaatgctgaatgggcttatgttttaaaagggtctactcaaatt

800 tctgctgttgataatgaagggagaaattatatttctactgttggtccagg

850 tgatttatggtattttccaccaggtattccacattctttacaagctactg

900ctgatgatccagaaggttctgaatttattttagtttttgattctggtgct

950 tttaatgatgatggtacttttttattaactgattggttatctcatgttcc

1000 aatggaagttattttaaaaaattttagagctaaaaatccagctgcttggt

1050ctcatattccagctcaacaattatatatttttccatctgaaccaccagct

1100 gataatcaaccagatccagtttctccacaagggactgttccattaccata

1150 ttcttttaatttttcttctgttgaaccaactcaatattctggtgggactg

1200 ctaaaattgctgattctactacttttaatatttctgttgctattgctgtt

1250 gctgaagttatactgttgaaccaggtgctttaagagaattacattggcatcc

1300 aactgaagatgaatggactttttttatttctggtaatgctagagttaacta

1350 tttttgctgctcaatctgttgcttctacttttgattatcaaggtggtgat

1400 attgctttatgttccagcttctatgggtcattatgttgaaaatattggtaa

1450 tactactttaacttatttagaagtttttaatactgatagatttgctgatg

1500tttctttatctcaatggttagctttaactccaccatctgttgttcaagct

1550 catttaaatttagatgatgaaactttagctgaattaaaacaatttgctac

1600 taaagctactgttgttggtccagttaattaa GCGGCCGC taaactat1646

Bacillus subtilis sequence (SEQ ID NO: 2): The underlined "G" at position 705 indicates an A→G base substitution. This base substitution does not change the amino acid sequence.

1 ATGAAAAAACAAAATGACATTCCGCAGCCAATTAGAGGAGACAAAGGAG

50 CAACGGTAAAAATCCCGCGCAATATTGAAAGAGACCGGCAAAACCCTGAT

100 ATGCTCGTTCCGCCTGAAACCGATCATGGCACCGTCAGCAATATGAAGTT

150TTCATTCTCTGATACTCATAACCGATTAGAAAAAGGCGGATATGCCCGGG

200 AAGTGACAGTACGTGAATTGCCGATTTCAGAAAACCTTGCATCCGTAAAT

250 ATGCGGCTGAAGCCAGGCCGATTCGCGAGCTTCACTGGCATAAAGAAGC

300 TGAATGGGCTTATATGATTTACGGAAGTGCAAGAGTCACAATTGTAGATG

350 AAAAAGGGCGCAGCTTTATTGACGATGTAGGTGAAGGAGACCTTTGGTAC

400 TTCCCGTCAGGCCTGCCGCACTCCATCCAAGCGCTGGAGGAGGGAGCTGA

450 GTTCCTGCTCGTGTTTGACGATGGATCATTCTCTGAAAACAGCACGTTCC

500 AGCTGACAGATTGGCTGGCCCACACTCCAAAAGAAGTCATTGCTGCGAAC

550 TTCGGCGTGACAAAAGAAGAGATTTCCAATTTGCCTGGCAAAGAAAAATA

600 TATATTTGAAAACCAACTTCCTGCCAGTTTAAAAGATGATATTGTGGAAG

650 GGCCGAATGGCGAAGTGCCTTATCCATTTACTTACCGCCTTCTTGAACAA

700 GAGCC G ATCGAATCTGAGGGAGGAAAAGTATACATTGCAGATTCGACAAA

750 CTTCAAAGTGTCTAAAACCATCGCATCAGCGCTCGTAACAGTAGAACCCG

800 GCGCCATGAGAGAACTGCACTGGCACCCGAATACCCACGAATGGCAATAC

850 TACATCTCCGGTAAAGCTAGAATGACCGTTTTTGCATCTGACGGCCATGC

900 CAGAACGTTTAATTACCAAGCCGGTGATGTCGGATATGTACCATTTGCAA

950 TGGGTCATTACGTTGAAAACATCGGGGATGAACCGCTTGTCTTTTTAGAA

1000 ATCTTCAAAGACGACCATTATGCTGATGTATCTTTAAACCAATGGCTTGC

1050CATGCTTCCTGAAACATTTGTTCAAGCGCACCTTGACTTGGGCAAAGACT

1100TTACTGATGTGCTTTCAAAAGAAAAGCACCCAGTAGTGAAAAAGAAATGC

1150 AGTAAATAA1158

The oxalate decarboxylase protein translated from the Bacillus subtilis sequence is shown below (Swiss-Prot: 034714) (SEQ ID NO: 3).

1 MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMK

50 FSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKE

100 AEWAYMIYGSARVTIVDEKGRSFIDVGEGDLWYFPSGLPPHSIQALEEGA

150 EFLLVFDDGSFSENSTFQLTDWLAHTPKEVIAANFGVTKEEISNLPGKEK

200 YIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPIESEGGKVYIADST

250 NFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGH

300 ARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWL

350 AMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK 385

Example 19. Stability of Soluble OXDC, Crystalline OXDC, and OXDC-CLEC at Low pH 3.0

2.5 mg/mL of soluble OXDC (Example 5), 5.0 mg/mL of crystalline OXDC (Example 5), and 5.0 mg/mL of OXDC-CLEC (Example 10) were placed in 1 mL of sodium citrate buffer, pH 3.0, in Eppendorf tubes and incubated at 37°C for 5 hours. Samples were taken at 0, 2, and 5 hours for enzyme stability measurement. Activity was determined as described in Example 15. The results showing the stability of OXDC and OXDC-CLEC at pH 3.0 after 0, 2, and 5 hours are shown in Figure 7. Compared to the initial incubation time, after 5 hours at pH 3.0, OXDC-CLEC retained approximately 100% activity, while soluble OXDC lost approximately 51% of its activity within the first 2 hours and only retained approximately 40% activity after 5 hours. Crystalline OXDC was more stable than soluble OXDC, retaining approximately 68% activity at 1 hour and approximately 67% activity at 2 hours.

Example 20. Stability of Soluble OXDC, Crystalline OXDC, and OXDC-CLEC Crystals in the Presence of Pepsin sex

1.0 mg/mL of soluble OXDC (Example 5), 10.0 mg/mL of crystalline OXDC (Example 5), and 1.0 mg/mL of OXDC-CLEC (Example 10) were placed in 1 mL of sodium citrate buffer, pH 3.0, in Eppendorf tubes and incubated for 5 hours at 37°C with pepsin (pepsin stock solution at a concentration of 1 mg/mL in 25 mM Tris-HCl buffer, pH 7.5) at a ratio of OXDC:pepsin of 50:1. Samples were taken at 0, 2, and 5 hours to measure enzyme stability. Activity was determined as described in Example 15. The results showing the stability of soluble OXDC, crystalline OXDC, and OXDC-CLEC in the presence of pepsin after 0, 2, and 5 hours are shown in Figure 8.

As shown in Figure 8, OXDC-CLEC preparations outperformed soluble OXDC at low pH and in the presence of pepsin. Compared to the initial activity, OXDC-CLEC retained approximately 60% activity after 5 h of incubation at 37°C, whereas most soluble OXDC and uncross-linked crystalline OXDC were degraded by pepsin after 2 h, retaining only approximately 20% activity after 5 h.

Example 21. Stability of Soluble OXDC, Crystalline OXDC, and OXDC-CLEC in the Presence of Chymotrypsin

1.0 mg/mL of soluble OXDC (Example 5), 10.0 mg/mL of crystalline OXDC (Example 5), and 1.0 mg/mL of OXDC-CLEC (Example 10) were placed in 1 mL of 25 mM Tris-HCl buffer, pH 7.5, in Eppendorf tubes and incubated with chymotrypsin (freshly prepared chymotrypsin stock solution at a concentration of 1 mg/mL in 25 mM Tris-HCl buffer, pH 7.5) at a ratio of OXDC:chymotrypsin of 50:1 at 37°C for 5 hours. Samples were taken at 0, 2, and 5 hours to measure enzyme stability. Activity was determined as described in Example 15. The results showing the stability of soluble OXDC, crystalline OXDC, and OXDC-CLEC in the presence of trypsin after 0, 2, and 5 hours are shown in Figure 9.

The results in Figure 9 demonstrate that after 5 hours of incubation at 37°C, OXDC-CLEC and uncrosslinked crystalline OXDC are stable and resistant to proteolytic cleavage by chymotrypsin. Analysis revealed that both OXDC-CLEC and uncrosslinked crystalline OXDC retained approximately 100% of their activity compared to their initial value 5 hours prior. Conversely, after 2 hours of exposure to chymotrypsin, the soluble protein lost approximately 50% of its activity, and after 5 hours, all soluble OXDC was degraded, leaving 0% activity.

Example 22. Stability of Soluble OXDC, Crystalline OXDC, and OXDC-CLEC in Simulated Intestinal Fluid Containing Pancreatin sex

5.0 mg/mL of soluble OXDC (Example 5), 10.0 mg/mL of crystalline OXDC (Example 5), and 10.0 mg/mL of OXDC-CLEC (Example 10) were placed in 1 mL of simulated intestinal fluid (prepared according to USB recommendations: 6.8 gm of potassium dihydrogen phosphate was dissolved in 250 mL of water, mixed with 77 mL of 0.2 N sodium hydroxide and 500 mL of deionized water; then, 10 gm of pancreatic enzyme was added, and the pH was adjusted to pH 6.8 with 0.2 N hydrochloric acid or 0.2 N sodium hydroxide; the volume was filled with water to 1 L) in Eppendorf tubes and incubated at 37°C for 2 hours. Samples were taken at 0, 1, and 2 hours for enzyme stability measurement. Activity was determined as described in Example 15. The results are shown in Figure 10.

The results shown in Figure 10 demonstrate that OXDC-CLEC is stable in simulated intestinal fluid containing pancreatic enzymes, maintaining approximately 100% activity. In contrast, most soluble OXDC is degraded by pancreatic enzymes (a mixture of lipase, amylase, and protease) within 1 hour, with only approximately 26%-28% activity remaining after 1 hour and 2 hours of incubation, respectively. In the presence of pancreatic enzymes, uncrosslinked crystalline OXDC is much more stable than its soluble form, retaining approximately 76% and 49% activity after 1 hour and 2 hours of incubation, respectively.

In addition, OXDC-CLEC protects the enzyme from cleavage by proteases such as pepsin and chymotrypsin. Under the same conditions, the stability of OXDC-CLEC can be at least about 100%, 200%, 300%, 400%, or more, of the stability of soluble OXDC. Under the same conditions, OXDC-CLEC maintains activity at least about 2, 3, or 4 times greater than the activity maintained by soluble OXDC. Thus, compared to soluble OXDC, OXDC-CLEC is active and stable under harsh intestinal conditions ranging from an acidic pH of about 2.5 or 3 to a pH of about 7.5 or 8.5, and in the presence of different proteases.

Many embodiments of the present invention have been described. Nevertheless, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the appended claims.

Claims (8)

1. The use of a capsule comprising spray-dried biologically active oxalate decarboxylase crystals and at least one excipient selected from sugars, sugar alcohols, thickeners, wetting agents, solubilizers, buffer salts, emulsifiers, antimicrobial agents, and antioxidants for oral administration in the preparation of a medicament for treating oxalate-related conditions in mammals, said oxalate-related conditions being selected from hyperoxaluria and nephrocalcification, wherein the water content of the spray-dried biologically active oxalate decarboxylase crystals is less than 5% by weight, provided that if the oxalate decarboxylase crystals are cross-linked, then the cross-linking of the oxalate decarboxylase crystals is carried out in the presence of 0.05% w/v to 2.0% w/v glutaraldehyde. 2. The application according to claim 1, wherein the hyperoxaluria is primary hyperoxaluria. 3. The application according to claim 1, wherein the hyperoxaluria is enterogenic hyperoxaluria. 4. The application according to any one of claims 1-3, wherein the oxalate decarboxylase crystals are uncrosslinked. 5. The application according to claim 1, wherein the crystal is active and stable in the gastrointestinal tract of mammals. 6. The application according to claim 1, wherein the crystal is active and stable at pH 2 to pH 8. 7. The application of claim 1, wherein treating oxalate-related conditions comprises reducing oxalate by at least 15%. 8. The application according to claim 1, wherein the oxalate decarboxylase crystals are cross-linked with 0.5% w/v glutaraldehyde.