CN101583375A - Crystallized oxalate decarboxylase and methods of use - Google Patents

Abstract

Oxalate decarboxylase crystals, including stabilized crystals, such as cross-linked crystals of oxalate decarboxylase, are disclosed. Methods to treat a disorder associated with elevated oxalate concentration using oxalate decarboxylase crystals are also disclosed. Additionally disclosed are methods of producing protein crystals.

Description

The oxalate decarboxylase of crystallization and using method

The cross reference of related application

The application requires the priority of U. S. application series number of submitting on August 2nd, 2,006 60/834,933 and the U. S. application series number of submitting on October 26th, 2,006 60/854,540, and their content is incorporated this paper into by reference in its entirety.

Background technology

Oxalic acid is formula HO ₂C-CO ₂The dicarboxylic acids of H.Oxalic acid is present in the organism mainly as oxalates, and described oxalates is the salt form of oxalic acid.Oxalates sees in the food, for example, and Herba Spinaciae, Radix Et Rhizoma Rhei, Fructus Fragariae Ananssae, Cranberries, nut, cocoa, chocolate, peanut butter, Sorghum vulgare Pers., and Folium Camelliae sinensis.Oxalates also is a metabolic end product in human and other mammal.It is advanced in the urine by renal excretion.When combining with calcium, oxalic acid generates insoluble product calcium oxalate, and it is the most general chemical compound of finding in renal calculus.

Because mammal can the synthesis and degradation oxalates enzyme, the levels of oxalate in the individuality is generally by draining and the low absorption of diet oxalates is kept normally.The oxalates of high concentration is relevant with multiple pathology, for example primary hyperoxaluria, intestinal originality hyperoxaluria and Te Fa hyperoxaluria.People such as Leumann, Nephrol.Dial.Transplant. 14: 2556-2558 (1999) and Earnest, Adv.Internal Medicine 24: 407-427 (1979).The reason of the oxalates that increases can be from the too much oxalates of food intake, too much to absorb the unusual of oxalates and oxalates generation from intestinal.Oxalates is relevant with enteropathy with the hyperabsorption in the small intestinal at colon, comprises the hyperabsorption that is caused by bile acid and the bad disease of fat absorption; Ileal resection; For example, the steatorrhea that causes by celiac disease, exocrine pancreas insufficiency, enteropathy and hepatopathy.

The urinary oxalate of oxaluria or increase is drained with many and at nephridial tissue (nephrocalcinosis) or urethra (is for example related to calcium oxalate, renal calculus (kidney stones), lithangiuria is with renal calculus (nephrolithiasis)) in sedimentary health problem relevant.Calcium oxalate also can be deposited on for example eyes, blood vessel, joint, bone, muscle, heart and other vitals, and they are caused damage.Referring to, for example, people such as Leumann, J.Am.Soc.Nephrol. 12: 19861993 (2001) and people such as Monico, Kidney International 62: 392400 (2002).The influence of the levels of oxalate that increases can appear in the multiple tissue.For example; deposition in little blood vessel can cause the skin ulcer of the pain that is difficult to fully recover; deposition in bone marrow can cause anemia, and the deposition in osseous tissue can cause fracture or influence child's growth, and the calcium oxalate deposition in heart can cause heart rate unusual or cardiac function is relatively poor.

The method of the levels of oxalate that existing treatment raises is always ineffective, and many primary hyperoxaluria patients may need intensive dialysis and organ transplantation.Different Hyperoxaluric existing therapies comprise the high dose vitamin B6, orthophosphate, magnesium, ferrum, aluminum, potassium citrate, colestyramine and glycosaminoglycans treatment, and the scheme of regulating diet and fluid absorption, dialysis and surgical operation (for example kidney and liver transplantation).These therapies (for example, low oxalates or low fat diet, vitamin B6, the enough calcium and the fluid of increase) only be that part is effective, they may have undesirable adverse side effect, for example orthophosphate, magnesium or the colestyramine gastrointestinal effects of replenishing and the risk of dialysis and operation.Therefore, need to remove from health safely the method for oxalates.In addition, the degraded oxalates reduces the method for the levels of oxalate in the biological sample, surpasses the therapy of removing such as the absorption or the acceleration oxalates of independent blocking-up oxalates.

Summary of the invention

The present invention relates to oxalate decarboxylase (" OXDC ") crystal and its cross-linked form (" CLEC ") and they and be used for the treatment of purposes such as Hyperoxaluric oxalates associated conditions.In one embodiment, the lenticular oxalate decarboxylase can be administered to mammal, and for example, per os or be applied directly to stomach deposits the infringement that causes to reduce levels of oxalate and/or to reduce calcium oxalate in mammal.In addition, the method for producing albumin crystal from cell extract is disclosed.The compositions that comprises oxalate decarboxylase (" OXDC ") crystal and its cross-linked form (" CLEC ") is also disclosed, for example, pharmaceutical composition.

In one aspect, the invention provides crosslinked oxalate decarboxylase crystal.Cross-linking agent can be multi-functional, and in certain embodiments, this reagent is bifunctional reagent, for example glutaraldehyde.In certain embodiments, oxalate decarboxylase crystal and the glutaraldehyde cross-linking that does not change concentration of enzymatic activity basically are for example at least about the concentration of 0.02% (w/v).In embodiments, the crystalline crosslinked level of oxalate decarboxylase equals to handle the level that produces with 0.02% (w/v) glutaraldehyde.Crosslinked level can be measured by method known in the art or disclosed herein, for example, measures the level of albumen leaching, and is for example disclosed as embodiment 10-11.

The present invention also provides the oxalate decarboxylase crystal, for example, has than soluble oxalate decarboxylase height for example at least about 100%, 200%, 300%, 400% or 500% active oxalate decarboxylase crystal.

The present invention also provide stabilisation, crosslinked oxalate decarboxylase crystal for example, active and/or the stability that the crystal of wherein said stabilisation keeps under acid condition is than at least 2,3 times of the active and/or stable height of soluble oxalate decarboxylase under similar acid condition (for example, about acid pH of 2 to 3).In embodiments, the oxalate decarboxylase crystal of described stabilisation is than the active and/or stable height at least 200%, 300%, 400% of the soluble oxalate decarboxylase under acid condition.

The present invention also provide stabilisation, crosslinked oxalate decarboxylase crystal for example, the crystal of wherein said stabilisation is at least 2,3 times of the active and/or stable height that has the active and/or stability that keeps in the presence of the protease to keep under conditions of similarity than soluble oxalate decarboxylase.In embodiments, the oxalate decarboxylase crystal of described stabilisation than soluble oxalate decarboxylase at the active and/or stable height at least 200%, 300%, 400% that has in the presence of the protease.Described protease can be selected from following one or more: for example, and pepsin, chymase or pancreatin.In embodiments, the crystal of described stabilisation or soluble oxalate decarboxylase are exposed to acid condition and/or protease preset time length for example at least 1,2,3,4 or 5 hours after, measure activity described stabilisation or soluble oxalate decarboxylase (for example, as this paper embodiment as described in).

One relevant aspect, the present invention has characterized a kind of crosslinked oxalate decarboxylase crystal, it under condition of different pH (for example, about pH 2.5 or 3 to 7.5 or 8.5) and/or having in the presence of the protease be activated basically and be stable, for example, protease can be selected from following one or more: for example, and pepsin, chymase or pancreatin.As described herein, in embodiments, the soluble oxalate decarboxylase of specific activity that described crosslinked crystal keeps (for example, about acid pH of 2 to 3) and at least 2,3 times of the active height of reservation in the presence of the protease are being arranged under acid condition.As described herein, in other embodiments, the oxalate decarboxylase crystal of described stabilisation than soluble oxalate decarboxylase at (for example, about acid pH of 2 to 3) under the acid condition with stable height at least 200%, 300%, 400% in the presence of the protease is being arranged.

Comprise crystal as herein described and/or the crosslinked crystalline compositions of oxalate decarboxylase, for example, pharmaceutical composition, also within the scope of the present invention.

In some embodiment, described crystal comprises the oxalate decarboxylase of the identical or substantially the same sequence of the sequence that has with the oxalate decarboxylase of finding in natural origin, and described natural origin is plant for example, antibacterial and fungus, especially bacillus subtilis, JINZHENGU or flammulina velutipes, aspergillus niger, Rhodopseudomonas (synechoystis sp.), cyanophyceae belongs to, Streptococcus mutans, hair bolt bacterium (Trameteshirsute), sclerotinite, whiterot fungi (T.versicolor), brown rot fungus (Postiaplacenta), myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria (Methylobacteriumextorquens), Pseudomonas oxalaticus really supports thunder Salmonella, Cupriavidusoxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and Mucor.In other embodiments, recombinant production oxalate decarboxylase.

In one aspect, the invention provides the method for the concentration of oxalate that reduces the experimenter, wherein use compositions disclosed herein, for example, pharmaceutical composition, it comprises the oxalate decarboxylase crystal, for example, crosslinked oxalate decarboxylase crystal.In one embodiment, described oxalate decarboxylase crystal is with cross-linking agent stabilisations such as glutaraldehydes.Using of compositions can make concentration of oxalate reduce at least 10%, at least 20%, at least 30% or at least 40% or more.In some embodiment, per os or by the device outside applying said compositions.In one embodiment, described device outside is a conduit, for example, scribbles the crystalline conduit of oxalate decarboxylase.In other embodiments, described compositions is used as suspension, dry powder, capsule or tablet.In one embodiment, the method for the mammiferous concentration of oxalate of described reduction comprises the step of measuring the concentration of oxalate in the mammiferous biological sample (for example urine, blood, blood plasma or blood serum sample).

In yet another aspect, the invention provides treatment, prevent and/or the slow down method of the progress of the disease relevant in the mammal with the concentration of oxalate that raises, wherein give described administration oxalate decarboxylase crystal and/or stabilisation, crosslinked oxalate decarboxylase crystal for example.In one embodiment, the described disease relevant with the concentration of oxalate that raises is nephropathy, arthrosis, oculopathy, hepatopathy, gastrointestinal disease or pancreas disease.In certain embodiments, described disease is a primary hyperoxaluria, intestinal originality oxaluria, and the special property sent out oxaluria, ethylene glycol is poisoned, cystic fibrosis, inflammatory bowel, lithangiuria, renal calculus, chronic nephropathy, hemodialysis and gastrointestinal bypass.

In yet another aspect, the invention provides a kind of compositions, for example, pharmaceutical composition, it comprises the oxalate decarboxylase crystal, for example, crosslinked oxalate decarboxylase crystal (for example, crystal disclosed herein and/or crosslinked crystal).

In yet another aspect, the invention provides the mammiferous method of treatment, wherein use the pharmaceutical composition of effective dose, the latter comprises the oxalate decarboxylase crystal, for example, and crosslinked oxalate decarboxylase crystal (for example, crystal disclosed herein and/or crosslinked crystal).

In yet another aspect, the invention provides and produce albumin crystal for example enzyme crystal is (for example, the oxalate decarboxylase crystal) method, it comprises: provide the goods that contain described proteic cell extract or granule/precipitation/solution and from the described albumen of described goods crystallization.In embodiments, described method comprises following one or more step: the described proteic prokaryotic host cell culture of culture expression; Obtain containing the granule of target protein or the goods of extract; The dissolved particles goods; Albumin crystal is formed, and/or by the crosslinked crystalcheckedization that further makes.Usually, recombinant expressed described albumen.In embodiments, particle product comprises inclusion body.

In embodiments, described dissolving step comprises, in particle product, add following one or more: the solution that comprises gentle denaturant concentration (for example, concentration is at about 1M for example extremely urea or the guanidine hydrochloride of about 3M); Comprise high salt concentration (for example, described salt is selected from one or more in sodium chloride, potassium chloride, the calcium chloride) or at for example solution of other salt of about concentration of 0.3 to about 0.8M; (for example, about 9 to about 12 pH) comprises the solution of gentle denaturant concentration under alkali condition; Or comprise the solution of high degeneration agent concentration (for example, about 4M to about 8M urea or guanidine hydrochloride).

In embodiments, described purification step comprises, from the particle removal fragment, for example, the particle product (for example, by one or more rotations or centrifugation step) by separate dissolvedization and/or collect supernatant.Described purification step can randomly also comprise, makes the particle product of dissolvingization pass ion exchange chromatography, and/or filters the goods of dissolvingization.

In embodiments, described crystallisation step comprises, concentrated and purified albumen, thereby the albumen of formation crystallization.In embodiments, the albumen of crystallization obtains from the supernatant of collecting behind separating step, for example, and after one or more rotations or centrifugation step.Crystallisation step can comprise in addition, makes the albumen contact cross-linking agent of crystallization, for example, and cross-linking agent disclosed herein (for example, glutaraldehyde).The concentration of the cross-linking agent that uses can be in 0.01% to 20%w/v scope; Common 0.02% to 10%w/v; More generally 0.02%, 0.5% or 1%w/v.

In embodiments, in the particle product proteic output be the specific protein in obtaining the cell product of particle product, found at least about 50%, 60%, 70%, 80%.In other embodiments, the proteic output of dissolvingization be in particle product, find at least about 90%, 95% or higher.In other embodiments, the proteic output of crystallization be in particle product, find at least about 50%, 60%, 70%, 80%.

The present invention also provides the albumin crystal of producing by method disclosed herein, for example, and enzyme crystal (for example, oxalate decarboxylase crystal).

In the the accompanying drawings and the following description, illustrated the details of one or more embodiments of the present invention.

Description of drawings

Fig. 1 is the figure that shows the pH living features of soluble oxalate decarboxylase (" soluble "), oxalate decarboxylase crystal (" crystal ") and crosslinked oxalate decarboxylase crystal (" CLEC ").

Fig. 2 describes the bar chart use the urinary oxalate level in the Sprague Dawley rat behind the OXDC-CLEC.Each bar is represented meansigma methods ± SE (standard error).Asterisk indication uses that two tail Student t checks calculate at the appointed time puts significant difference p＜0.05 between matched group and 3 the treatment groups.

Fig. 3 is a bar chart of describing the influence of the reduction of urinary oxalate level in (KO) mice that AGT1 that OXDC-CLEC attacks ethylene glycol knocks out.Each bar is represented meansigma methods ± SE.Asterisk indication uses that two tail Student t checks calculate at the appointed time puts significant difference p＜0.05 between matched group and 3 the treatment groups.

Fig. 4 describes the bar chart of OXDC-CLEC to the influence of the creatinine clearance in the AGT1KO mice of ethylene glycol attack.Each bar is represented meansigma methods ± SE.The matched group of two tail Student t check calculating and significant difference p＜0.05 between the 80mg treatment group are used in the asterisk indication.

Fig. 5 A-5C shows with OXDC-CLEC to treat the metanephros image of the sedimental prevention of calcium oxalate in fact.The use by oneself section of excess of the kidney matter of the mice that EG that 80mg OXDC-CLEC handles attacks of Fig. 5 A.Fig. 5 B and 5C are the sections from the excess of the kidney matter of matched group.The section of Fig. 5 B confirms medium nephrocalcinosis, and the section of Fig. 5 C confirms serious nephrocalcinosis.Dark speckle is calcium oxalate deposit (example of pointing out with white arrow), and light speckle is zone with interstitial fibrosis (example of pointing out with grey arrow).

Fig. 6 is the Kaplan-Meier survival figure of contrast with the time-to-live of the mice of the EG attack of the OXDC-CLEC of 3 kinds of various dose or medium control treatment.

Fig. 7 be described in designated time intervals, the figure of the stability of soluble oxalate decarboxylase (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ") when low pH.

Fig. 8 be described in designated time intervals, when pH 3.0, at the figure of the stability that soluble oxalate decarboxylase in the presence of the pepsin (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ") are arranged.

Fig. 9 be described in designated time intervals, when pH 7.5, at the figure of the stability that soluble oxalate decarboxylase in the presence of the chymase (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ") are arranged.

Figure 10 be described in designated time intervals, when pH 6.8, at the figure of the stability that soluble oxalate decarboxylase in the presence of the simulated intestinal fluid that contains pancreatin (" Sol "), oxalate decarboxylase crystal (" XTAL ") and crosslinked oxalate decarboxylase crystal (" CLEC ") are arranged.

Detailed Description Of The Invention

The present invention is based in part on following discovery, uses oxalate decarboxylase (OXDC) crystal and can alleviate mammiferous hyperoxaluria symptom. This paper describes the method that the OXDC crystal is treated different oxalates associated conditions of using. In addition, provide OXDC crystal and crosslinked crystal (CLECs), comprise and use their composition. In addition, the method for producing a large amount of albumin crystals from the cell extract of prokaryotic host cell is disclosed.

Definition.In order more easily to understand the present invention, at first define some term. In detailed description, set forth other definition.

" biological sample " used herein is the biomaterial of collecting from cell, tissue, organ or organism, for example, and for detection of analyte. Exemplary biological sample comprises liquid, cell or tissue sample. Biological fluids comprises, for example, and serum, blood, blood plasma, saliva, urine or sweat. The cell or tissue sample comprises biopsy, tissue, cell suspending liquid, or other sample and sample, for example clinical sample.

" crystal " is a kind of form of solid matter, its comprise the atom that the pattern that repeats with three-dimensional periodic arranges (referring to, for example, Barret, Structure of Metals, 2^ndEd., McGraw-Hill, New York (1952)). The crystal form of polypeptide for example, is different from the second form-amorphous solid. Crystal shows unique feature, comprises shape, lattice structure, percentage of solvents and optical property, for example, and refractive index.

" device outside " is in vivo structure not, is used for making body fluid contact OXDC crystal in the treatment of individuality. Preferably, device outside is the device be used to the dialysis that comprises the kidney dialysis, is used for the device of CAVH, ECMO, or be used for from other device of blood flow filtration refuse. Similarly, this term comprises the assembly of the device that filters refuse, comprises for example conduit, porous material or film. More specifically, device outside can be dialysis apparatus. It also can be the film of dialysis apparatus.

" function fragment " of OXDC is a part that has kept one or more bioactive OXDC polypeptide of OXDC, and described activity is the ability of the decarboxylation of catalysis oxalates for example. Function fragment used herein can comprise the terminal butt from one or two end, except as otherwise noted. For example, function fragment can have from the amino of OXDC polypeptide and/or carboxyl terminal abridged 1,2,4,5,6,8,10,12,15, or 20 or more residue. Preferably, butt is no more than 20 amino acid from one or two end. Function fragment can randomly be connected to one or more heterologous sequence.

Term " individuality " or " experimenter " refer to any mammal, include but not limited to, any animal of classification comprises the people like this, non-human primate, primate, baboon, orangutan, monkey, rodent (for example, mouse, rat), rabbit, cat, dog, horse, ox, sheep, goat, pig, etc.

Term " separation " refers to basically to break away from the molecule of its natural surroundings. For example, the albumen of separation breaks away from cell material or basically from its other albumen in cell or tissue source in source. This term refers to that the purity of the albumen that wherein separates is enough to the goods of using as therapeutic combination, or at least 70% to 80% (w/w) purity, more preferably at least 80% to 90% (w/w) purity, 90 to 95% purity more preferably; At least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) purity most preferably.

Term " about " used herein refers to be up to the value that this term limits ± 10%. For example, about 50mM refers to 50mM ± 5mM; About 4% refers to 4% ± 0.4%.

" oxalates associated conditions " used herein refers to disease or the illness relevant with the pathology level of oxalic acid or oxalates, including, but not limited to, hyperoxaluria, primary hyperoxaluria, intestines originality hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol (oxalates) is poisoned, the idiopathic urinary stone disease, kidney failure (comprise gradual, chronic or latter stage kidney failure), stearrhea, malabsorption, ileum is sick, Vulvodynia, cardiac conduction obstacle (cardiac conductance disorders), inflammatory bowel disease, cystic fibrosis, EPI, Crohn disease, ulcerative colitis, nephrocalcinosis, lithangiuria, and kidney stone. Such illness and obstacle can randomly be acute or chronic. The oxalates associated conditions relevant with kidney, bone, liver, intestines and stomach and pancreas known in the art. In addition, well-known, calcium oxalate can deposit in Various Tissues, includes, but not limited to eyes, blood vessel, joint, bone, muscle, heart and causes other vitals of many oxalates associated conditions.

PH (pK at urine and intestinal juice_a1＝1.23，pK _a2=4.19), " oxalic acid " mainly exists with its salt form oxalates (as the salt of the conjugate base of correspondence). Earnest, Adv.Internal Medicine24: 407427 (1979). Term " oxalic acid " and " oxalates " be Alternate in this disclosure. The oxalates that comprises lithium, sodium, potassium and iron (II) is soluble, (for example, only is dissolved to about 0.58mg/100ml at 18 ℃ but calcium oxalate is insoluble in water usually very much. Earnest, Adv.Internal Medicine24: 407427 (1979)). Oxalic acid from food is also referred to as the diet oxalates. The oxalates that is produced by metabolic process is called the endogenous oxalates. The circulation oxalates is to be present in for example oxalates in the blood of instrument for circulation of body fluid.

Term " treatment effective dose " or " treatment effective dose " refer to, the amount of compound that causes the improvement of the delay of the prevention of (comprising hyperoxaluria, for example primary hyperoxaluria or intestines originality hyperoxaluria) of oxalates associated conditions, paresthesia epilepsy or symptom. The treatment effective dose be enough to for example to treat, prevent, alleviate the illness relevant with the concentration of oxalate that raises seriousness, postpone its outbreak and/or reduce the outbreak of one or more symptoms dangerous. By the well-known in the art and described method of further part this specification, can determine effective dose.

Term " treatment ", " methods for the treatment of " and their near synonym refer to (prophylatic)/prevention (preventative) measure that treats and/or prevents of existing illness. Need the object for the treatment of can comprise the individuality that has had the certain medical illness and be in the danger of described illness or have described illness or may final ill object. Following evaluation is to the demand for the treatment of: for example, by the existence of the one or more hazards relevant with advancing of disease, the existence of illness or progress, or to the possible acceptance of the treatment of experimenter with described illness. Treatment can comprise the progress that slows down or reverse illness.

Oxalate decarboxylase.Oxalate decarboxylase used herein (OXDC) (EC 4.1.1.2) refers to the oxalic acid carboxy-lyase. Oxalate decarboxylase is known in the artly can be independent of molecular oxygen (O according to following reaction₂) catalysis oxalic acid be oxidized to one group of enzyme of carbon dioxide and formic acid:

HO ₂C-CO ₂H→1CO ₂+HCOOH

The sugared shape of the isotype of oxalate decarboxylase and those isotypes is included in this definition. This term comprises the OXDC from plant, bacterium and fungi, comprise the true oxalate decarboxylase from bacterium and fungi, bacillus subtilis for example, Asparagus or flammulina velutipes, aspergillus niger, pseudomonas, blue-green algae belongs to (Synechocystis sp.), streptococcus mutans, hair bolt bacterium (Trametes hirsute), sclerotinite, whiterot fungi (T.versicolor), brown rot fungus (Postia placenta), myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria (Methylobacterium extorquens), Pseudomonas oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and Mucor. Randomly, OXDC can depend on coacetylase in addition, for example from the OXDC of enteron aisle biology. In some cases, OXDC is soluble six glycoprotein polyprotein precursors.

Oxalate decarboxylase is generated by higher plant, bacterium and fungi, and has oxalic acid carboxy-lyase activity. Oxalate decarboxylase comprises by following biological those that generate: bacillus subtilis, Asparagus or flammulina velutipes, aspergillus niger, pseudomonas, blue-green algae belongs to (Synechocystis sp.), streptococcus mutans, hair bolt bacterium (Trametes hirsute), sclerotinite, whiterot fungi (T. versicolor), brown rot fungus (Postia placenta), myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria (Methylobacterium extorquens), Pseudomonas oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and Mucor, they are usually differentiated and are cupin type OXDC. Cupin-sample albumen is the albumen of total some architectural feature of a large class. OXDC is activated from the G-OXDC that Collybia belongs to as for example six aggressiveness glycoprotein for example.

Can separate from for example natural origin with the oxalate decarboxylase that is used for methods described herein for the preparation of crystal, maybe can be derived from natural origin. Term used herein " is derived from " and refers to have naturally occurring amino acid or nucleotide sequence in the source. For example, the oxalate decarboxylase that is derived from bacillus subtilis comprises the basic sequence of bacillus subtilis oxalate decarboxylase albumen, or by the nucleic acid coding of the sequence that is included in the coding oxalate decarboxylase found in the bacillus subtilis or its degradation product. The albumen or the nucleic acid that are derived from a source comprise molecule that separate from this source, recombinant production and/or chemical synthesis or that modify. Crystal provided herein can be from the amino acid sequence that comprises OXDC or the polypeptide preparation that keeps the OXDC function fragment of oxalates degrading activity. Preferably, OXDC keeps at least one functional character of naturally occurring OXDC, for example, keep following one or more: ability, multimerization ability and/or the manganese demand of catalysis oxalates degraded.

The oxalate decarboxylase that separates.Isolated in the past oxalate decarboxylase, thereby can obtain from many sources, comprise bacillus subtilis, Asparagus or flammulina velutipes, aspergillus niger, pseudomonas, blue-green algae belongs to (Synechocystis sp.), streptococcus mutans, hair bolt bacterium (Trametes hirsute), sclerotinite, whiterot fungi (T.versicolor), brown rot fungus (Postia placenta), myrothecium verrucaria, Agaricus bisporus, methylotrophic bacteria (Methylobacterium extorquens), Pseudomonas oxalaticus, Ralstonia eutropha, Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Variovorax paradoxus, xanthobacter autotrophicus, Eurotium, Penicillium, and Mucor. OXDC also can be available from commercial supplier, for example, and Sigma. The method of separating OXDC from natural origin once was described in, for example, and in the following list of references: the people such as Tanner, The Journal of Biological Chemistry.47: 43627-43634. (2001); Dashek, W.V.and Micales, J.A., Methods in plant biochemistry and molecular biology. Boca Raton, FL:CRC Press.5:49-71. (1997); The people such as Magro, FEMS Microbiology Letters.49:49-52. (1988); The people such as Anand, Biochemistry.41：7659-7669.(2002)；and Tanner and Bornemann，S.Journal of Bacteriology. 182: 5271-5273 (2000). The oxalate decarboxylase of these separation can be used to form crystal as herein described and method.

The oxalate decarboxylase of reorganization.Perhaps, the OXDC of reorganization can be used to form crystal provided herein and method.In some cases, the OXDC of reorganization comprises the sequence from naturally occurring OXDC sequence, or by its coding.In addition, this paper has described the OXDC that comprises with naturally occurring sequence homology or substantially the same aminoacid sequence.Also provide by with the OXDC of naturally occurring OXDC-homologous coding nucleic acid or substantially the same nucleic acid coding, and can crystallization as described herein, and/or use.

Be called " reorganization " polypeptide in this article and be the polypeptide that has generated, comprise by depending on artificial recombination for example polymerase chain reaction (PCR) and/or use restriction endonuclease to clone those that the into operation of the method for carrier produces by recombinant DNA method." reorganization " polypeptide also is the polypeptide expressed with change, for example has the naturally occurring polypeptide of the expression of recombinant modified in cell (for example host cell).

In one embodiment, OXDC be from bacillus subtilis or the homologous nucleic acid recombinant production of JINZHENGU OXDC nucleotide sequence, and modify sometimes, for example, to increase or to optimize the recombinant production in heterologous host.An example of the sequence of this modification is provided in SEQ ID NO:1 (nucleic acid), and it comprises the nucleotide sequence of the opening code-reading frame of JINZHENGU OXDC, is used for expressing at Candida boidinii.Described OXDC sequence is modified to be connected to α mating factor secretory signal sequence to reduce its GC content with it, and the restriction endonuclease cleavage site of side joint through engineering approaches.In another embodiment, OXDC is the bacillus subtilis OXDC nucleotide sequence recombinant production from the SEQ ID NO:2 or the unmodified that can obtain at GenBankAccession No:Z99120.Be provided as SEQ ID NO:3 by SEQ ID NO:2 amino acid sequence coded.

Can be used for forming the crystalline OXDC polypeptide of OXDC can express in host cell, for example comprises the host cell of nucleic acid construct, and described nucleic acid construct contains the coded sequence of OXDC polypeptide or its function fragment.The host cell that is fit to expression OXDC can be yeast, antibacterial, fungus, insecticide, plant or mammalian cell, for example, or transgenic plant, transgenic animal or acellular system.Preferably, host cell is glycosylation OXDC polypeptide where necessary, can form disulfide bond, can secrete OXDC, and/or can support the multimerization of OXDC polypeptide.Preferred host cell including, but not limited to, escherichia coli (comprising escherichia coli Origami B and e. coli bl21), pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces pombe, bacillus subtilis, aspergillosis, Sf9 cell, Chinese hamster ovary (CHO), 293 cells (human embryo kidney) and other people's cell.Transgenic plant, transgenic animal (comprising pig, cattle, goat, horse, chicken and rabbit) also are the hosts who is fit to produce OXDC.

For recombinant production OXDC, host or host cell should comprise construct, the latter be comprise at least one coding OXDC or its function fragment nucleic acid plasmid, carrier, phasmid or transcribe or the form of expression cassette.Can obtain many kinds of constructs, comprise the construct of keeping with single copy or a plurality of copy, or mix the construct in the host cell chromosome.Many recombinant expression systems, component and recombinant expressed reagent can commercially obtain, for example from Invitrogen Corporation (Carlsbad, CA); U.S.Biological (Swampscott, MA); BD BiosciencesPharmingen (San Diego, CA); Novagen (Madison, WI); Stratagene (LaJolla, CA); And Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), (Braunschweig, Germany).

OXDC's is recombinant expressed randomly by allogeneic promoter, comprises the control of composing type and/or inducible promoter.Promoter for example, T7, alcohol oxidase (AOX) promoter, dihydroxy-acetone synthase (DAS) promoter, Gal 1,10 promoteres, phosphoglyceric kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, the alcoholdehydrogenase promoter, copper metallothionein (CUP1) promoter, the acid phosphatase promoter, CMV and promoter polyhedrin also suit.Based on host or host cell, select concrete promoter.In addition, for example, can also can use, and be well-known in the art by methanol, copper sulfate, galactose, hypophosphate, the inductive promoter of alcohol (for example, ethanol).

The nucleic acid of coding OXDC can randomly comprise heterologous sequence.For example, in some embodiment, secretion sequence is included in the N-end of OXDC polypeptide.Can use signal sequence, for example from those of α mating factor, BGL2, leavening acid acid phosphatase (PHO), xylanase, alpha amylase, from other yeast secretary proteic those and be derived from the secreting signal peptide of excretory other kind that can instruct host cell.Similarly, for example junctional complex is (for example for other heterologous sequence, it comprises cutting or restriction endonuclease site) and one or more expression control element, enhancer, terminator, leader and one or more translation signals, all in the scope of this description.These sequences can randomly be included in the construct and/or be connected on the nucleic acid of coding OXDC.Except as otherwise noted, " connection " sequence can directly or indirectly combine each other.

Similarly, for example histidine, HA (hemagglutinin peptide), maltose-binding protein, AviTag of epi-position or affinity tag

, FLAG or glutathione-S-transferase can randomly be connected on the OXDC polypeptide.Behind production or purification, can randomly cut label down from OXDC.The technical staff can easily select the heterologous sequence that suits, for example, and coupling host cell, construct, promoter, and/or secretory signal sequence.

OXDC homologue or variant and OXDC canonical sequence differ one or more residues.For example can be with similar some specified aminoacid of amino acid replacement on the structure.Similar aminoacid comprises on the structure: (I, L and V); (F and Y); (K and R); (Q and N); (D and E); (G and A).OXDC homologue as herein described also comprises amino acid whose disappearance, interpolation or displacement.Such homologue and variant comprise: (i) polymorphie variant and natural or artificial mutant, (ii) modified polypeptides has wherein been modified one or more residues and has (iii) been comprised the mutant of the residue of one or more modifications.

If it and canonical sequence have at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homogeneity, then OXDC polypeptide or nucleic acid are " homologous " (or " homologue ").If homologue is different with canonical sequence, then it is " variant ".If the nucleotide of homologue or aminoacid sequence and canonical sequence (for example differ, by truncate, disappearance, displacement or interpolation) be no more than 1,2,3,4,5,8,10,20 or 50 residue and keep the ability (or coding keeps polypeptide of this ability) of catalysis oxalates degraded, then homologue with reference to OXDC sequence " substantially the same ".The fragment of oxalate decarboxylase can be homologue, comprises variant and/or substantially the same sequence.As an example, homologue can be derived from different OXDC sources, or they can be derived from canonical sequence or relevant with canonical sequence by truncate, disappearance, displacement or interpolation sudden change.Homogeneity percentage ratio between 2 nucleotide or the aminoacid sequence can be measured by the alignment algorithm of standard, for example, people such as Altschul, J.Mol.Biol., 215: the Basic Local Alignment Tool (BLAST) described in 403410 (1990), people such as Needleman, J.Mol.Biol., 48: 444453 (1970) algorithm, or people such as Meyers, Comput.Appl.Biosci. 4: 11 17 (1988) algorithm.Such algorithm mixes BLASTN, and (summary is seen McGinnis and Madden, Nucleic Acids Res. for BLASTP and " BLAST 2 Sequences " program 32: W20-W25,2004).When using such program, can use default parameters.For example, for nucleotide sequence, following setting can be used for " BLAST 2Sequences ": program BLASTN, mate 2 awards (reward for match 2), 2 point penalties of mispairing (penalty for mismatch 2), it is respectively 5 and 2 that point penalty (extension gap penalties) is extended in room open (open gap) and room, gap x_dropoff50, expect 10, word size 11, filter ON.For aminoacid sequence, following setting can be used for " BLAST 2 Sequences ": program BLASTP, matrix B LOSUM62, it is respectively 11 and 1 that point penalty (extension gap penalties) is extended in room open (open gap) and room, gap x_dropoff 50, expect 10, word size 3, filter ON.The aminoacid and the nucleotide sequence that are fit to formation crystalline OXDC described herein can comprise homologous, variant or substantially the same sequence.

The purification of oxalate decarboxylase.Before crystallization, can be from (source for example natural or reorganization) purification oxalate decarboxylase albumen or the polypeptide of originating.Being called " isolating " polypeptide in this article is, (for example, their origin (for example to break away from its natural surroundings basically, cell, tissue (that is, plant tissue) albumen, lipid and/or nucleic acid, or liquid or culture medium (under the situation of secrete polypeptide))) polypeptide.Isolating polypeptide comprises those that obtain by methods described herein or other appropriate method, comprises pure in fact or pure basically polypeptide and by chemosynthesis, polypeptide by recombinant production or the combinations produce by the biological and chemical method.Randomly, isolating albumen through further processing, for example passes through purification step after production.

Purification can comprise buffer exchange and chromatographic step.Randomly, can use concentration step, for example, by dialysis, chromatofocusing chromatography, and/or binding buffer fluid exchange.In some cases, cation or anion-exchange chromatography are used for purification, comprise the Q-agarose, DEAE agarose, DE52, sulfopropyl Sepharose Chromatography or CM52 or similar cation exchange column.Buffer exchange randomly before chromatographic isolation, and can by grossflow filtration for example diafiltration realize.In some goods, OXDC is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% purity.

Be applicable to preparation OXDC with the purification that carries out of gram magnitude, be effective, cheap, production scale OXDC purification optimization operation.The purification of at least 0.5,1,2,5,10,20,50,100,500 or 1000 grams or more OXDC for example, is provided in purification process.In an exemplary operation, provide 10L, 50L, 100L, 500L, 1000L or more initial sample grossflow filtration at least, to realize the precipitation of buffer exchange and contaminating protein.Single Q-agarose column randomly is used for purification OXDC.

The crystallization of the OXDC of purification also can be removed pollutant, for example is further purified the OXDC goods.For example, compare the low-molecular-weight pollutant that have the reduction level as the OXDC of crystallization as described in the embodiment 2-6 with the OXDC of soluble purification.Aspect some, optionally get rid of from crystal form and to have 0-10KDa, 1-10KDa, 0.5-5KDa, or the pollutant of the measurement quality of 2-5KDa (by the auxiliary laser desorption ionisation mass spectral analysis of substrate (MALDI-MS)).For example, by crystallization, can remove basically and have about 2.5,3.0,3.7,3.8,4.0,4.2 or the pollutant of the measurement quality of 5.0KDa.Use the fermentation medium that for example contains thick oxalate decarboxylase, also can crystallization purifying.

The crystallization of oxalate decarboxylase.Use for example six aggressiveness of above-mentioned OXDC polypeptide, can prepare the oxalate decarboxylase crystal (referring to people such as Anand, Biochemistry 41: 7659-7669 (2002)).Can use for example diffusion of vapor (for example, hanging drop and seat drip a method) and batch crystallization method.By making albumen from aqueous solution or comprise controlled crystallization the aqueous solution of organic solvent, can form the oxalate decarboxylase crystal gradually.The condition that can control comprises for example existence, pH and the temperature of evaporation rate, suitable cosolute and the buffer agent of solvent.

Use for treatment, for example be used for the treatment of disease relevant with levels of oxalate or obstacle, multiple OXDC crystal size suits.In certain embodiments, use crystal less than about 500 μ m average-sizes.Average, the maximum with about 0.01,0.1,1,5,10,25,50,100,200,300,400,500 or 1000 μ m length or the oxalate decarboxylase crystal of minimum dimension (for example) also are provided.Crystallite showers also is suitable.

Scope suits, and the technical staff can understand.For example, albumin crystal can have the longest dimension of about 0.01 μ m to about 500 μ m or 0.1 μ m to about 50 μ m.In a specific embodiments, the longest dimension scope is that about 0.1 μ m is to about 10 μ m.Crystal also can have the shape that is selected from ball, pin, rod, plate, for example hexagon and square, rhombus, cube, bipyramid and prism.In explanatory embodiment, crystal is the cube that has less than the longest dimension of 5 μ m.

Generally speaking, by will crystalline albumen with suitable aqueous solvent or to contain the aqueous solvent of suitable crystallizing agent (for example salt or organic solvent) combined, produce crystal.Make described solvent and albumen combined, and randomly be fit to induced crystallization and keep protein active and the stirring of stable acceptable temperature experience in measuring.Solvent can randomly comprise cosolute, for example unit price or bivalent cation, cofactor or chaotropic agent, and the buffer agent of control pH.Measuring promotes required cosolute of crystallization and their concentration.In the commercial scale process,, can produce crystalline controlled precipitation by for example in batch process, making albumen, precipitant, cosolute and optional buffer combined.Can adopt substituting laboratory method for crystallising and condition, for example (McPherson waits the people, Methods Enzymol. for dialysis or diffusion of vapor 114: 112-20 (1985) andGilliland, Crystal Growth 90: 51-59 (1998)).Once in a while, the incompatibility between cross-linking agent and the crystallization medium may change buffer (solvent) before crosslinked.

As described in embodiment, oxalate decarboxylase can crystallization under many conditions, comprises wide pH scope (for example, pH 3.5-8.0).In described some embodiment, comprise precipitant for example Polyethylene Glycol (for example, PEG 200, and PEG 400, PEG 600, and PEG 1000, and PEG 2000, PEG 3000, and PEG 8000[is referring to embodiment 7 and 8]) or organic cosolvent 2-methyl-2 for example, 4-pentanediol (MPD).Operable ordinary salt comprises sodium chloride, potassium chloride, ammonium sulfate, zinc acetate etc.

The concentration of oxalate decarboxylase in the crystallization fermentation liquid can be, for example, and at least 5,10,15,20,25,30,35,40,45,50,60,70,80,90, or 100mg/ml, or higher.The efficient of crystallization reaction or productive rate are at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.In one embodiment, make oxalate decarboxylase solution and suitably cushion liquid-phase mixing, form or produce the oxalate decarboxylase crystal gradually by batch process.In certain embodiments, described buffer is 100mM Tris-HCl pH of buffer 8.0 and the 100mM NaCl that contains 2mM cysteine-HCl.

From cell or cell extract crystallization.Crystal can be directly from cell or the preparation of granular cell extract.In one embodiment, results are expressed the bacterial cell of oxalate decarboxylase.Be with or without in the presence of the DNA enzyme suspension cell again, and homogenization.Saline solution is added cell lysate, reach about 0.3M, 0.4M, 0.5M, 0.6M or higher salinity.The salt that adds can be sodium salt, potassium salt, calcium salt or other salt.By removing cell debris, can randomly extract albumen from cell mixture.In one embodiment, the cell mixture of homogenization is centrifugal, make albumen in supernatant soln.By reducing the salinity of cell mixture or protein solution, produce crystal.In one embodiment, remove salt by dialysis, to keep protein concentration.In order to improve crystal yield, can be before the salinity that reduces solution protein concentrate solution.Can in about 6,7 or 8 the solution of pH, produce crystal.

Can be from albumen precipitation thing or preparation of granules crystal.In one embodiment, results are expressed the cell of target protein, and collect oxalate decarboxylase albumen in precipitate or granule.To contain proteic granule of oxalate decarboxylase or precipitate and be dissolved in saline solution.By reducing the salinity of protein solution, form crystal.In order to improve crystal yield, before reduction concentration was produced crystal, the salinity in the dissolved protein solution was at least about 0.3M, 0.4M, 0.5M or higher.

Also can prepare crystal from protein solution.In one embodiment, in saline solution, concentrate the oxalate decarboxylase protein solution, when reducing the salinity of solution, form crystal.In order to improve crystal yield, before reduction concentration was produced crystal, salinity was at least about 0.3M, 0.4M, 0.5M or higher.

The crystal of stabilisation.In case the oxalate decarboxylase crystal forms in suitable medium gradually, can randomly make their stabilisations, for example by crosslinked.By between crystalline component protein molecule, introducing covalent bond, the crosslinked stabilisation that causes lattice.This make albumen is transferred to otherwise may be incompatible with existing of described lattice or even with existing of intact proteins inconsistent alternative environment in become possibility.The oxalate decarboxylase crystal can be by for example lysine amido, sulfydryl (sulfydryl) and carbohydrate partly come crosslinked.Crosslinked crystal is also referred to as " OXDC-CLEC, " " CLEC-OXDC, " or " CLEC " in this article.

Crosslinked crystal can change enzyme stability (for example, pH, temperature, machinery and/or chemical stability), the active pH character of OXDC, dissolubility, the homogeneity of crystal size or volume, the speed that enzyme discharges from crystal, and/or hole size and shape between each enzyme molecule in basic lattice.

Advantageously, show at least 60%, 80%, 100%, 150%, 200%, 250%, 300% or the mode of more highly active OXDC, carry out crosslinked or stabilisation according to the present invention so that crystal comprises to compare with soluble OXDC.Compare with soluble OXDC, stability can improve at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more.Can under condition of storage, measure stability, pH stability for example, temperature stability, the stability of anti-erepsin, biological stability in stripping stability and the body, for example.

In some embodiment, the crosslinked meeting stripping of OXDC polypeptide in solution in the crystal of slowing down is immobilized in protein molecular in the microcrystal grain effectively.After the triggering agent in the surrounding that is exposed to crosslinked albumin crystal, for example under use rather than condition of storage, protein molecular is dissolving slowly, discharges active OXDC polypeptide and/or increases the OXDC activity.Can control dissolution rate, for example one or more by in the following factor: the degree of cross linking, albumin crystal are exposed to the time span of cross-linking agent, the speed that in albumin crystal, adds cross-linking agent, the character of cross-linking agent, the chain length of cross-linking agent, pH, temperature, the existence of thiol (sulfahydryl) reagent (as cysteine, glutathion), the surface area of crosslinked albumin crystal, the shape of the size of crosslinked albumin crystal and crosslinked albumin crystal.

A kind of in crosslinked (abreast) at the same time or the multiple cross-linking agent that uses in order or their combination realize, comprise multi-functional dose.After the triggering agent in being exposed to surrounding, or through given time phase, and crosslinked between the crosslinked albumin crystal of such polyfunctional crosslinking agent diminish or die down, and causes albumen stripping or active release.Perhaps, crosslinked can the fracture at binding site causes albumen stripping or active release.Referring to U.S. Patent number 5,976,529 and 6,140,475.

In some embodiment, cross-linking agent is to have at least 2,3,4,5, or polyfunctional crosslinking agent of more a plurality of active parts.In different embodiments, described reagent can be selected from glutaraldehyde, butanedial, suberic aldehyde, Biformyl, two sulfur two (succinyl phosphorons amino propyl acid ester), 3,3 ' two sulfur two (thiosuccimide base propionic ester), 3, the two third imidic acid dimethyl ester HCl of 3 '-dithio, N-succinimido-3-(2-pyridine radicals two sulfur) propionic ester, cyclohexanediamine, the diaminourea octane, ethylenediamine, succinic anhydrides, the phenyl glutaric anhydride, salicylide, acetimide ester (acetimidate), formalin, acrylic aldehyde, the succinum semialdehyde, butyraldehyde, lauryl aldehyde, glyceraldehyde and trans-oct-2-ene aldehyde.

Other polyfunctional crosslinking agent comprises the halo triazine, for example, and cyanuric chloride; Halogenated pyrimidine, for example, 2,4,6-trichlorine/bromo pyrimi piperidine; The anhydride of aliphatic series or aromatic monocarboxylate or dicarboxylic acids or halogenide, for example, maleic anhydride, (methyl) acryloyl chloride, chloracetyl chloride; The N-methylol compound, for example, N-methylol chloro-acetamide; Vulcabond or diisothio-cyanate, for example, phenylene-1,4-vulcabond and aziridine.Other cross-linking agent comprises epoxide, for example, and diepoxide, triepoxides and Fourth Ring oxide.In one embodiment, cross-linking agent is a glutaraldehyde, and promptly a kind of difunctional dose, glutaraldehyde uses separately or uses in order with epoxide.Also can with reversible cross-linking agent (for example following those) (abreast) or other cross-linking agent that uses in order (referring to, for example, the catalogues in 1996 of Pierce Chemical Company) simultaneously.

According to an alternative embodiment of the present invention, can be abreast or sequentially to use reversible cross-linking agent to carry out crosslinked.The crosslinked albumin crystal that obtains is characterised in that reactive multi-functional cross-linking agent, wherein triggers thing and mixes as separating group.Reactive functionality participates in the acid of the reactive amino in albumen side chain is connected together, and triggers thing by coming destructive key to form by the one or more conditions (for example, pH, the existence of Reducing agent, temperature, or thermodynamics water activity) that change in the surrounding.

Cross-linking agent can be congenerous or exclusive-OR function.Reactive functionality (or part) can, for example, be selected from one of following functional group (R wherein, R ', R ", and R " ' can be alkyl, aryl or hydrogen group):

I. reactive acry radical donor, for example: carboxylate RCOOR ', amide RCONHR ', acyl azide RCON ₃, carbodiimide R-N=C=N-R ', N hydroxyl imide ester, RCO-O-NR ', imines ester R-C=NH2 ⁺(OR '), anhydride RCO-O-COR ', carbonic ester RO-CO-O-R ', urethanes RNHCONHR ', acid halogenide RCOHal (wherein Hal=halogen), acid hydrazide RCONNR ' R " and (O-acyl group isourea) OAcylisoureas RCO-O-C=NR ' (NR " R " ');

II. reactive carbonyl, for example: aldehyde RCHO and ketone RCOR ', acetal RCO (H ₂) R ' and ketal RR ' CO2R ' R " (at document (Pierce Catalog and Handbook, PierceChemical Company, Rockford, Ill. (1994); S.S.Wong, Chemistry ofProtein Conjugation and Cross-linking, CRC Press, Boca Raton has described the functional group of containing reactive carbonyl known to the skilled in proteopexyization and crosslinked field among the Fla. (1991));

III. alkyl or aryl donor, for example: alkyl or aryl halogenide R-Hal, azide R-N ₃, sulfuric ester RSO ₃R ', phosphate ester RPO (OR ' ₃), alkyl oxonium salt R ₃O+, sulfonium R ₃S+, nitrate RONO ₂, Michael receptor RCR '=CR " ' COR ", aryl fluoride ArF, isonitrile RN+=C-, halogenated amine R ₂N-Hal, alkene, and alkynes;

IV. sulfur-containing group, for example: disulphide RSSR ', sulfydryl RSH and epoxide R ₂C_ ⁰CR ' ₂With

V. salt, for example: alkyl or aryl ammonium salt R ₄N+, carboxylate RCOO-, sulfate ROSO ₃-, phosphate ROPO ₃" and amine R ₃N.

Reversible cross-linking agent for example, comprises the triggering thing.Trigger thing and comprise alkyl, aryl, or have can with other chain of the activated group of wanting crosslinked albumino reaction.Those reactive groups can be the groups of any kind, for example are easy to those of displacement of nucleophilic displacement, free radical or electrophilic displacement, comprise halogenide, aldehyde, carbonic ester, urethanes, xanthane and epoxide and other.For example, reactive group can be to acid, alkali, fluoride, enzyme, reduction, oxidation, sulfydryl, metal, photolysis, root (radical) or thermally labile.

At T.W.Green, Protective Groups in Organic Synthesis, JohnWiley﹠amp; Other example of reversible cross-linking agent has been described among the Sons (Eds.) (1981).The strategy that is used for any kind of reversible protecting group can be mixed and be fit to produce the cross-linking agent that to realize the crosslinked protein crystal of reversible controlled dissolution.Different schemes is listed in the AngewanteChemie Inl.Ed.Engl. of Waldmann, 35: in 2056 (1996) the summaries about this theme.

The reversible cross-linking agent of other type is to contain the disulfide bond crosslinking agent.Destroying the triggering thing that is cross-linked to form by such cross-linking agent is to add Reducing agent in the environment of crosslinked albumin crystal, for example cysteine.In Pierce Catalog and Handbook (1994-1995), exemplary disulphide cross-linking agent has been described.At U.S. Patent number 6,541, the such cross-linking agent and the example of method are disclosed in 606, its relevant portion is by with reference to introducing.

In addition, also can use between carbohydrate part or crosslinked cross-linking agent between carbohydrate part and aminoacid.

The concentration of cross-linking agent solution can be about 0.01% to 20%, about 0.02% to 10% or about 0.05% to 5%w/v.Usually, cross-linking agent is about 0.5% or about 1%w/v.For example, the concentration of 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[referring to the table 2 among the embodiment].May be before crosslinked exchange buffering liquid.Randomly lyophilized or otherwise prepare crystal comprises CLEC.

Crystal comprises crosslinked crystal as herein described, can be used for the method for Therapeutic Method as herein described and reduction levels of oxalate.The OXDC crystal also can be used for (for example, synthesizing processing, bioreediation with industrial process, sterilization, sterilization) method of relevant method and treatment plant (for example mycotic infection of plant), for example summarize referring to, for example, people such as Svedruzic, Arch.Biochem.Biophys. 433: 176-192 (2005).So non-therapeutic use of soluble or unbodied OXDC referring to, for example, U.S. Patent number 5,866,778; 6,218,134; 6,229,065; 6,235,530; With 6,503,507.Based on crystalline one or more character of the OXDC of aforementioned stableization, crystal as herein described can be used for these purposes, for example increases the stability of oxalate decarboxylase.

The crystalline drying of oxalate decarboxylaseRemove water, organic solvent or liquid polymers by following manner, thereby dry oxalate decarboxylase crystal: comprise, with nitrogen, air or noble gas drying, the vacuum drying oven drying, lyophilized, with volatile organic solvent washing, evaporating solvent subsequently, in fume hood, evaporate the tray drying, fluid bed drying, spray drying, vacuum drying, or cylinder dry.Usually, when crystal becomes free flowable powder, carry out drying.By making gas stream pass wet crystal, can carry out drying.Described gas can be selected from: nitrogen, argon, helium, carbon dioxide, air or their combination.

In principle, can prepare exsiccant crystal by lyophilized.But this technology comprises the quick cooling of material, is only applicable to the stable product of lyophilizing.In one embodiment, the aqueous solution that at first will contain the lenticular oxalate decarboxylase is freezing to-40 to-50 ℃, takes out under vacuum then.

Oxalate decarboxylase crystal or comprise the so crystalline preparation or the production of compositions.In one aspect, oxalate decarboxylase crystal or comprise such crystalline preparation or compositions is disclosed.Such compositions can prepare according to following method:

At first, make the oxalate decarboxylase crystallization.Then, excipient or the composition that is selected from sugar, sugar alcohol, viscosifier, wetting agent or solubilizing agent, buffer salt, emulsifying agent, antimicrobial, antioxidant and coating materials directly added mother solution.Perhaps, remove mother solution, then crystal was suspended in excipient solution minimum 1 hour to maximum 24 hours.Excipient concentration normally about 0.01 is to about 10% (w/w).Described constituent concentration is about 0.01 to about 90% (w/w).Described crystal concentration is about 0.01 to about 99% (w/w).

Then by filtering or, removing mother solution from crystal slurry by centrifugal.Subsequently, randomly in room temperature or making an appointment with-20 ℃ of extremely about 25 ℃ temperature, with about 50-100% (w/w) solution washing crystal of one or more organic solvents (for example, ethanol, methanol, isopropyl alcohol or ethyl acetate).

Then by making nitrogen, air or inert gas flow pass through them, dried crystals.Perhaps, by air drying, spray drying, lyophilized or vacuum drying, dried crystals.After the washing, dry minimum about 1 hour to about 72 hours at most, up to the water content of end-product be lower than about 10% (by weight), more preferably less than about 5% (by weight).At last, can carry out crystalline littleization (reducing size) if desired.

According to one embodiment of the invention, when preparing the oxalate decarboxylase crystal or comprising so crystalline preparation or compositions, in crystallization process, do not add reinforcing agent, for example surfactant.After crystallization described excipient or composition are added in the mother solution, its concentration is about 1 to about 10% (w/w), and perhaps concentration is about 0.1 to about 25% (w/w), and perhaps concentration is about 0.1 to about 50% (w/w).To about 3 hours, perhaps incubation about 0.1 was to about 12 hours with the crystal incubation in described excipient or composition and the mother solution about 0.1, and perhaps incubation about 0.1 was to about 24 hours.

In another embodiment of the invention, described composition or excipient are dissolved in the mother solution solution in addition, take out crystal from mother solution, be suspended in excipient or the composition solution.Described composition or excipient concentration and incubation time are identical with above-mentioned those.

Another advantage of the present invention is, can come drying to be encapsulated in polymer by lyophilized and carry intravital oxalate decarboxylase crystal or its preparation comprise microsphere with formation compositions.Lyophilized or lyophilization allow to separate water outlet from compositions.At first freezing oxalate decarboxylase crystalline composition places fine vacuum then.In a vacuum, the water of crystallization distillation, remaining oxalate decarboxylase crystalline composition, it only contains the water of combining closely.The trade union that adds like this makes the further stabilisation of compositions, and make in the storage of common ambient temperature and transportation easier.

Spray drying allows to separate water outlet from crystalline articles.But it is highly suitable for the drying solid of continuous production powder, granule or agglomerate form from the liquid charging stock of the form of suspension of solution, emulsion and pump.Spray drying comprises, and liquid charging stock is atomized into droplet spray, and makes microdroplet contact hot-air in hothouse.Generate spraying by rotary (wheeled) or nozzle-type aerosol apparatus.Under controlled air temperature and current condition, moisture evaporates from microdroplet, forms dried granules.The temperature that the spray drying action need is higher relatively.But, mostly just be slight to the pyrolytic damage of product, this be because the evaporative cooling effect in the crucial drying stage and because dry matter to be exposed to the pyritous time subsequently very short.Powder is discharged continuously from hothouse.According to the dry feature and the powder specification of product, selection operation condition and exsiccator design.Spray drying is a kind of ideal process, and wherein end-product must meet accurate quality standard aspect particle size distribution, residual moisture content, bulk density and the grain shape.

Compositions.The OXDC crystal comprises crosslinked crystal, as compositions for example pharmaceutical composition (referring to, for example, U.S. Patent number 6,541,606, it has described the preparation and the compositions of albumin crystal) provide.Comprise the crystalline pharmaceutical composition of OXDC and comprise OXDC crystal and one or more compositions or excipient, include, but are not limited to sugar and biocompatible polymer.The case description of excipient is in the Handbook of Pharmaceutical Excipients of American Pharmaceutical Association (American Pharmaceutical Association) and Britain pharmaceutical society (Pharmaceutical Society of Great Britain) combined publication, and other example is as described below.

The OXDC enzyme can be used as in the multiple physiologically acceptable salt form any and uses as crystal in compositions, and/or with acceptable pharmaceutical carrier and/or additive as part of pharmaceutical compositions.The medicine preparation technique of physiologically acceptable salt form and standard and excipient be those skilled in the art well-known (referring to, for example, Physician ' s DeskReference (PDR) 2003,57th ed., Medical Economics Company, 2002; With Remington:The Science and Practice of Pharmacy, compile people such as .Gennado, 20th ed, Lippincott, Williams﹠amp; Wilkins, 2000).For application purpose, " preparation " comprises " crystal formulations ".

The oxalate decarboxylase that can be used in the inventive method can be combined with excipient.According to the present invention, the filler combination that " excipient " is used as filler or uses in pharmaceutical composition.The exemplary composition and the excipient that are used for compositions are as described below.

Biocompatible polymerBiocompatible polymer is nonantigenic (when not being when the adjuvant), non-carcinogenic, nontoxic and additionally they are not and live organism incompatible polymers inherently, and they can be used for OXDC crystalline composition as herein described.Example comprises: poly-(acrylic acid), poly-(cyanoacrylate), poly-(aminoacid), poly-(anhydride), poly-(depsipeptide), poly-(ester) for example poly-(lactic acid) or PLA, poly-(lactic acid-copolymerization-glycolic) or PLGA, poly-(beta-hydroxy-butanoic acid ester), poly-(caprolactone) and poly-(dioxanone); Poly-(ethylene glycol), poly-((hydroxypropyl) isobutyl Methacrylamide, poly-[(organic) phosphine nitrile], poly-(adjacent ester), poly-(vinyl alcohol), poly-(vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether co-polymer, Pluronic polyhydric alcohol (pluronic polyols), albumin, alginate esters, cellulose and cellulose derivative, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharide, glucosaminoglycan, sulfated polysaccharides, their admixture and copolymer.

Biodegradable polymeric, promptly the polymer that can degrade by hydrolysis or dissolving can be included in the OXDC crystalline composition.Degraded can be heterogeneous (mainly occurring in particle surface) or homogeneity (the whole polymeric matrix of degrading equably).

Can in the OXDC crystalline composition, comprise composition such as one or more excipient or ingredient or excipient.Composition can be inert or activated composition.

Method with OXDC crystal treatment oxalates associated conditionsMethod of the present invention comprises, uses oxalate decarboxylase to mammalian subject, and for example OXDC crystal or its cross-linked form are with treatment, prevention or reduce causing danger of the disease relevant with the levels of oxalate of rising.Can detect the levels of oxalate that raises in for example from experimenter's biological sample, for example body fluid comprises urine, blood, serum or blood plasma.In certain embodiments, detect the urinary oxalate level.In method as herein described, can use crystal and/or comprise crystalline compositions.

In some embodiment, provide treatment to have oxaluria, the special Hyperoxaluric method of sending out the individuality of property oxaluria, oxalosis that primary hyperoxaluria, intestinal originality oxaluria, surgical operation cause.In other cases, the oxalates associated conditions of the rising of kidney, bone, liver, gastrointestinal tract and pancreas can be suitable for treatment disclosed herein.Other disease by method provided herein treatment or disease including, but not limited to, ethylene glycol (oxalates) is poisoned, the special property sent out urinary stone disease, renal failure (comprise gradual, chronic or latter stage renal failure), steatorrhea, malabsorption, the ileum disease, vulvodynia, cardiac conduction obstacle (cardiac conductance disorders), inflammatory bowel, cystic fibrosis, EPI, Crohn disease, ulcerative colitis, nephrocalcinosis, osteoporosis, lithangiuria, and renal calculus.It is acute or chronic that such disease and obstacle can be chosen wantonly.

Method of the present invention can make the levels of oxalate among the experimenter compare reduction at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more with the level among untreated or the contrast experimenter.In some embodiment,, measure and reduce by contrast experimenter's levels of oxalate before using OXDC and afterwards.In some embodiment, the invention provides the method for the treatment of or improving oxalates associated conditions or obstacle, so that one or more symptoms of described disease or obstacle improve at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.In certain embodiments, described method reduces the level of endogenous oxalates and/or the absorption of diet oxalates.

In some embodiment, provide treatment to have the method for the genotypic individuality relevant with high levels of oxalate, for example reduce the sudden change individuality that isozygoty or heterozygosis of following enzymatic activity: for example, alanine: glyoxalic acid aminotransferase, glyoxylate reductase/hydroxypyruvate reductase, liver oxyacetate oxidase, or participate in oxalates metabolism or the another kind of enzyme relevant with oxaluria.In other embodiments, provide treatment to have method reduction or that lack the individuality of Oxalobacter formigenes intestinal cluster.

Disclosed method comprises, gives susceptible, suffers from the disease relevant with the levels of oxalate of rising or is in the oxalate decarboxylase of the mammalian subject administering therapeutic effective dose in this danger.Colony by method of the present invention treatment including, but not limited to, suffer oxalates associated conditions (for example, primary hyperoxaluria or intestinal originality oxaluria) or be in experimenter in the danger of this disease of development.

The experimenter of the method according to this invention treatment comprises the people, non-human primate, primate, baboon, orangutan, monkey including, but not limited to mammal, rodent (for example, mice, rat), rabbit, cat, Canis familiaris L., horse, cattle, sheep, goat, pig, etc.

Indication, symptom and disease indication.Many methods can be used to pass judgment on the development or the progress of oxalates associated conditions or the disease relevant with the levels of oxalate that raises.Such disease including, but not limited to, arbitrarily disease, disease or obstacle as defined above.By for example measuring urinary oxalate, blood plasma oxalates, measure kidney or liver function, or detect the calcium oxalate deposit, can pass judgment on the development or the progress of oxalates associated conditions.

By detecting or measure the concentration of oxalate in urine sample for example or other biological sample or the liquid, can differentiate disease, disease or obstacle.Hyperoxaluric early symptom is renal calculus normally, may be with stomachache serious or burst or hypochondriac pain, hematuria, frequent urgent micturition, the pain of urinating or heating and shiver with cold.Renal calculus can be Symptomatic or asymptomatic, and can observe by abdominal part X ray for example, ultrasonic or computerized tomography (CT) scanning imagery.If do not control oxaluria, kidney can be impaired, and impaired renal function.Kidney even possibility are depleted.Minimizing by urinary volume or lack (glomerular filtration rate), general ill sensation, tired and significant fatigue, feel sick, vomiting, anemia and/or be difficult to can differentiate renal failure (and poor kidney) in child's normal development in period and growth.By direct range estimation (for example using eyes), X ray, ultrasonic, CT, ultrasoundcardiogram or biopsy methods such as (for example, bone, liver or kidney), also can detect the calcium oxalate deposit in other tissue and organ.

Use art-recognized direct and indirect determination method, also can pass judgment on kidney and liver function and concentration of oxalate.By well-known technology, also can test compounds content or urine, blood or other biological sample.For example, can measure oxalates, oxyacetate and glycerate level.The algoscopy of liver and renal function is well-known, for example, analyzes the oxalates deposit of the azymia and the analysis nephridial tissue of hepatic tissue.The DNA that causes primary hyperoxaluria that notifies that also can specimen changes.

Other treatment indication including, but not limited to, have one or more risk factor, comprise front and those of part discussion below.Whether existence by finding out one or more such risk factor, diagnosis or prognostic indicator, can differentiate the experimenter who is in development or susceptible disease, disease or obstacle or may especially easily accept the experimenter of oxalate decarboxylase treatment.Similarly, by analyzing one or more genotypies or phenotype mark, can differentiate the individuality in the danger that is in development oxalates associated conditions.

The urinary oxalate level that disclosed method can be used for per 24 hours periods 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 400mg oxalates or more experimenter.In certain embodiments, levels of oxalate is relevant with one or more symptoms or condition of illness.Can measure the levels of oxalate in the biological sample, for example body fluid comprises blood, serum, blood plasma or urine.Randomly, oxalates is standardized into the albumen or the material of standard, for example the kreatinin in the urine.In some embodiment; claimed method comprises; use oxalate decarboxylase; in 1,3,5,7,9,12 or 15 day; the subject's circulating levels of oxalate is reduced to the level that can not detect, or be reduced to the treatment before experimenter's levels of oxalate less than 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.

The human Hyperoxaluric urinary oxalate of every day that is characterised in that is drained greater than 40mg (about 440 μ mol) or 30mg.Exemplary clinical cutoff level is, for example, the male is 43mg/ days (about 475 μ mol), and the women is 32mg/ days (about 350 μ mol).Oxaluria also can be defined as the urinary oxalate of every gram urine creatine acid anhydride every day and drain greater than 30mg.Slight oxaluria patient can drain 30-60 (342-684 μ mol) or 40-60 (456-684 μ mol) mg oxalates at least every day.Intestinal originality oxaluria patient can drain 80mg urinary oxalate (912 μ mol) at least every day, the primary hyperoxaluria patient can drain 200mg (2280 μ mol) at least every day, for example, Borowski A.E, Langman CB.Hyperoxaluria and Oxalosis:Current Therapy and Future directions.Exp Opinion Phrama (2006, in the printing).

Using of OXDC crystal and its compositions

Using of oxalate decarboxylase according to the inventive method is not limited to any specific drug-supplying system, comprise by upper gastrointestinal for example mouth (for example in capsule, suspension, tablet, or with food) or the top of stomach or intestinal (for example by conduit or injection) administration, to reduce individual levels of oxalate.In some cases, use OXDC and reduce endogenous levels of oxalate and/or concentration.OXDC also can be provided by device outside, dialysis apparatus for example, and conduit, or contact is from the structure or the device of the biological sample of individuality.

Individual administration can occur in single dose or the repeat administration, and with in the multiple physiologically acceptable form any, and/or use acceptable pharmaceutical carrier and/or additive as part of pharmaceutical compositions (aforementioned).In method disclosed by the invention, oxalate decarboxylase can be individually, with one or more other bioactivators side by side or through the administration continuously of eclipsed or nonoverlapping interval, described other bioactivator for example, pyrrole many hot (vitamin B-6s), orthophosphate, magnesium, glycosaminoglycans, calcium, ferrum, aluminum, magnesium, potassium citrate, colestyramine, organic marine products hydrocolloid, plant juice, for example, Fructus Musae stem juice or beet juice, or L-cysteine.Provide and reduced levels of oxalate or increase the activity of OXDC or the bioactivator of availability.In successive administration, oxalate decarboxylase and one or more other reagent can be with any order administrations.In some embodiment, the length of section gap can be to surpass for 2,4,6,12,24 or 48 weeks or more.

Oxalate decarboxylase can be used as unique reactive compound or with the ground administration of another kind of reactive compound or combination of compositions.Except as otherwise noted, according to the progress oxalate decarboxylase of the seriousness of symptom and disease dosed administration with about 10 μ g/kg to 25mg/kg or 100mg/kg.The suitable treatment effective dose of OXDC is selected by the treatment clinicist, its approximate range is 10 μ g/kg to 20mg/kg, 10 μ g/kg to 10mg/kg, 10 μ g/kg to 1mg/kg, 10 μ g/kg to 100 μ g/kg, 100 μ g/kg to 1mg/kg, 100 μ g/kg to 10mg/kg, 500 μ g/kg to 5mg/kg, 500 μ g/kg to 20mg/kg, 1mg/kg to 5mg/kg, 1mg/kg to 25mg/kg, 5mg/kg to 100mg/kg, 5mg/kg to 50mg/kg, 5mg/kg to 25mg/kg and 10mg/kg to 25mg/kg.In addition, can use in an embodiment or 57th ed., Medical Economics Company, the concrete dosage of pointing out in 2002 at Physician ' s DeskReference (PDR) 2003.

Oxalate decarboxylase crystal of the present invention can be by for example being delivered to oxalate decarboxylase patient's device outside or catheter drug delivery.Conduit, for example, catheter can wrap and be contained the crystalline compositions of oxalate decarboxylase.

The following examples provide explanatory embodiment of the present invention.Those of ordinary skills can understand many improvement and the variation that can make under the situation that does not change the spirit or scope of the present invention.Such improvement and variation are within the scope of the present invention.Embodiment does not limit the present invention in any way.

Embodiment

The fermentation and the purification of embodiment 1. oxalate decarboxylases.

261kDa homotype six glycoprotein polyprotein precursors of forming by 6 identical monomers from the oxalate decarboxylase (OXDC) of bacillus subtilis (B.subtilis).Each monomer contains 385 aminoacid, and the molecular weight of calculating is～43-44kDa that isoelectric point, IP is 5.2.Use the bacillus subtilis genomic DNA as template, pcr amplification was called the OXDC gene of yvrK in the past.

(CA), the e. coli bl21 (DE3) of sub-clone, and use then pLysS cell is expressed from the pET-11a expression vector for Invitrogen, Carlsbad at first the OXDC gene clone of amplification to be advanced the pCRII carrier.This gene expression in the pET-11a carrier is under the control of T7 promoter, by inducing with IPTG (isopropyl-), regulates OXDC and expresses.

Use fermentation to realize the high level expression of OXDC in escherichia coli of recombinating.Containing casein hydrolysate (USB Corporation, Cleveland, OH) or soy peptone, yeast extract (USB Corporation), NaCl (Fisher Scientific), PPG 2000 defoamer (PPG), KOH (Mallinckrodt Baker, Inc., Phillipsburg, NJ), and in 800L (liter) fermentation medium of ampicillin (USB Corporation) express.Because OXDC is manganese-dependent enzyme, comprises 5mM MnCl in fermentation medium ₂4H ₂O (Mallinckrodt Baker, Inc.).By adding 0.4mM IPTG (Lab Scientific), the expression of inducing OXDC.The cell of expressing OXDC is grown in and shakes in bottle or the fermentation tank.By this method, OXDC mainly is expressed in the preparation of granules thing.

To use the glycerol original seed of the BL21 cell of pET11a-OXDC conversion to be used for fermentation.For the 800L fermentation, prepare the preceding culture of seed with 2 * 250ml flask, each flask contains 50ml culture medium (LB+100 μ g/ml ampicillin).Use 2 * 0.5ml inoculum.Cultivated culture 6 hours at 35 ℃, 250rpm.Then this culture is transferred to 6 * 2L flask, each flask contains 1L culture medium (LB+100 μ g/ml ampicillin).In each flask, add the 10ml starting culture.Cultivated these cultures 12 hours at 35 ℃, 250rpm.This culture of 6L is transferred to 800L to be contained in the fermentation tank of above-mentioned appropriate culture medium.Culture is grown in 37 ℃, pH 7.0, shakes at 100rpm that to make dissolved oxygen be about 30-40, up to OD ₆₀₀Reach about 0.3 (the about 2-3 of this process need hour).With 0.4mM IPTG+5mM MnCl ₂4H ₂O, the expression of inducing OXDC.37 ℃, 100rpm inducing culture thing 4 hours.Harvesting then, and freezing standby.OD when results ₆₀₀Be about 5.5-8.0.

Be with or without in the presence of the 15-25U/ml DNA enzyme, containing the ratio of the buffer of 50mMTris pH 8,100mM NaCl, suspension cell again with 1kg cell paste/4L.Spend the night at 4 ℃ of mixing (50-60rpm) cell suspending liquid.Make cell suspending liquid be passed on ice pre-cooled homogenizer 3 times.In test under microscope lysis efficient, with not broken cell suspending liquid with comparing.4 ℃, in the 1L bottle, 4,000rpm centrifuge cell 40min.Preserve supernatant and granule, be used for SDS-PAGE and characterize.By SDS-PAGE, analyze the expression of OXDC.Find most OXDC in granule, it comprises inclusion body and other precipitation.By at the centrifugal 40min of 4000rpm, the results granule uses it or freezing standby at-70 ℃ immediately.

From the 800L fermentation reaction, we obtain 5,000 to 5, the 500g cell, and they produce 2,800 to 3,000g weight in wet base granule.

Gentle denaturant concentration of embodiment 2. usefulness and anion-exchange chromatography subsequently are from dissolvingization Granule crystallization oxalate decarboxylase.

To be used to prepare the OXDC crystal at the OXDC of-20 ℃ of cold preservations granule.

In this operation, dissolved particles under the denaturant concentration of temperate condition and pH.Use anion exchange substrate post then, again the albumen of folding dissolvingization.

Granule is dissolved in 2M urea, 100mM Tris pH 10.0,10mM DTT and 100mMNaCl (1: 10w/v).At room temperature (RT) agitating solution 2h, then at 4 ℃ at 15K centrifugal solution 30min.Supernatant decanted liquid, and preservation carefully.Carefully granule is weighed preservation respectively.

Under constant and soft stirring, with the flow velocity of 10ml/min, with the granule of the dissolvingization in the supernatant dropwise add 10 volumes by 2M urea, 100mM Tris pH 8.0,1mM DTT and 1mM MnCl ₂In the solution of forming.After adding the supernatant end, at room temperature incubation protein solution 1h.Behind the incubation, by 4 ℃, at the centrifugal 30min of 15K, to remove arbitrarily possible precipitation, for anion-exchange chromatography is ready to protein solution.

By the Q agarose matrix being packed in the glass column preparation anion-exchange column.Described post is connected on the FPLC, by 0.5M urea, 100mM Tris pH8.0,1mM DTT and the 1mM MnCl with 10 column volumes (CV) ₂Solution washing carries out balance.Flow velocity maintains 6ml/min.The granule of dissolvingization is loaded upper prop.Protein load is a 8-10mg/ml substrate.After the load sample, at the flow velocity of 6ml/min, with 100mM Tris pH 8.0,1mM DTT and the 1mM MnCl of at least 10 column volumes ₂Column scrubber.This step is removed urea from bonded protein sample, and makes albumen be folded into its native conformation again.

By discontinuous gradient or stepwise elution, use 1M NaCl, 100mM Tris pH 8.0 and 1mM DTT from the post eluted protein.Monitor the albumen eluting at 280nm, collect the 10ml fraction.The peak fraction is merged together, tests by SDS-PAGE and enzymatic activity.

The following peak fraction that will contain OXDC is concentrated into 15mg/ml: use 10, the 000MWCO film stirs cell, dialyses in the 100mM of 10 volumes Tris pH 8.0,100mM NaCl and 1mM DTT.With 1 hour at interval, change buffer 2 times, change buffer for the third time after, continue dialysed overnight, to realize the maximum crystal response rate.By 4 ℃, at the centrifugal sample 15min of 2K, be recovered in crystalline albumen in the bag filter.After the dialysis, the OXDC of about 70% refolding can crystallization.Crystal is a cube shaped, has the homogeneous size.Crystal shows the activity of about 44 units.

Embodiment 3. uses high degeneration agent concentration and anion-exchange chromatography subsequently, by dissolving Granule makes the oxalate decarboxylase crystallization.

By this method, granule is dissolved in 5M urea, 50mM Tris pH 8.6,100mM NaCl, 10mM DTT (1: 5w/v).At stirring at room solution 2h, then 4 ℃, at 15K centrifugal solution 30min.Supernatant decanted liquid, and preservation carefully.Carefully granule is weighed preservation respectively.

By the Q agarose matrix being packed in the glass column preparation anion-exchange column.Described post is connected on the FPLC, by 4M urea, 100mM Tris pH 8.6 and the 10mM DTT washing with 3 column volumes (CV), carries out balance.100mM NaCl, 50mM TrispH 8,1mM MnCl with 7 column volumes ₂, 10mM DTT, further column scrubber, in single step with 0.5M NaCl, 50mM Tris pH 8.0,1mM DTT, the 1mM MnCl of 3 column volumes ₂Eluting.

Collect suitable fraction, identify albumen by SDS-PAGE and determination of activity.Having in the presence of the high salt (0.5M NaCl), concentrating soluble albumen.With 0.5M eluting salt 450ml cumulative volume, to 45ml, enter 100mM NaCl, 50mM Tris pH 8.0,1mM DTT through the pellicon filtering and concentrating.Albumen dilutes, so that make salinity be reduced to 0.1MNaCl from 0.5M after concentrating.At this moment, the OXDC crystal begins to form.In this embodiment, in 100mM NaCl, 50mM Tris pH 8.0,1mM DTT equivalent, volume is transferred to 210mL.The crystal that rotation forms is being with or without in the presence of the 1mM DTT, reclaims in 100mM NaCl and 50mM Tris pH8.0.

Embodiment 4. uses high pH and gentle denaturant concentration, carry out doughnut subsequently concentrates, Make the oxalate decarboxylase crystallization by dissolved particles.

In room temperature, will contain inclusion body and other sedimentary granule (3.93kg) and be dissolved in 9.5L 50mMTris pH 12,500mM NaCl, 2M urea, 10mM DTT 2 hours.Final volume is 12L, and pH 9.9.7000rpm rotary sample 45 minutes, reclaim supernatant.Postrotational cumulative volume is 11.1L, and pH 9.9.On doughnut with sample concentration to 5L1 hour, use 50mM Tris pH 8.0,500mM NaCl, 2M urea, 10mM DTT then, volume is slowly transferred to 20L.This process was carried out 1 hour.At this moment, the final concentration of estimation urea is about 0.5M urea.In doughnut, volume is concentrated into 6L 2.5h.With 50mM Tris pH 8.0,500mM NaCl, 1mM DTT, 1mM MnCl ₂, 200mM L-arginine, with diluted sample to 24L 30min.At this moment, the concentration of estimation urea is 125mM.Carry out another take turns concentrated, to final volume 5L.With 50mM Tris pH 8.0,500mM NaCl, 1mM DTT, 1mM MnCl ₂, diluted sample to 18L, is concentrated into 6.5L once more.This moment, pH was 8.1.7000rpm rotary sample 45 minutes, preserve final granule and be used for analyzing.After centrifugal, carry out dilution step, obtain crystal with 50mM Tris pH 8.0,1mM DTT.In room temperature, under mixing, use peristaltic pump to be diluted to 30L.The flow velocity of estimating diluent is 50ml/min.This dilution was carried out about 9 hours.By centrifugal collection crystal, the preservation supernatant is used for analyzing.With 50mM Tris, 100mM NaCl pH8 washing crystal 3 times, be suspended in 50mM Tris, 100mM NaCl pH 8 again.At 4 ℃ of preservation crystal.

Embodiment 5. uses high salt to make oxalate decarboxylase crystallization from cell extract

(1) Use the high salt concentration dissolving to contain proteic granule, concentrate subsequently and dilute, tie Brilliant

In room temperature, the frozen particle (465g) of embodiment 1 is dissolved among 2.3L 100mM Tris, 1mM L-cysteine HCL, 0.5M NaCl, the pH 8.0 2 hours, form soluble oxalate decarboxylase.7000rpm rotary sample 45 minutes, reclaim supernatant.Final volume is 2.15L, and the protein concentration that records is 24.14mg/ml.By grossflow filtration (10kD Pall), sample concentration to 550ml 1 hour, at the flow velocity of 73ml/min, was diluted to 2 through 30 minutes with 100mM Tris pH 8.0,1mM L-cysteine HCl then, 750L was stirring at room 1 hour.The formation crystal spends the night in the cold house.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM Tris, 100mM NaCl pH 8 washing crystals 3 times, be suspended in 100mM Tris, 100mM NaCl pH 8 then again.At 4 ℃ of preservation crystal.Under standard test condition (referring to embodiment 15), show the ratio work of about 50-60U/mg from the recombined bacillus subtilis OXDC of escherichia coli expression culture medium purification.

(2) Use the high salt concentration dissolved particles, concentrate subsequently and dialyse, carry out crystallization

In room temperature, the frozen particle (510g) of embodiment 1 is dissolved among 3L 100mM Tris, 1mML-cysteine HCL, 0.5M NaCl, the pH 8.0 2 hours.7000rpm rotary sample 30 minutes, reclaim supernatant.By grossflow filtration (10kD Pall), with sample concentration to 500ml 1 hour, dialysis in 100mM Tris pH 8.0 under agitation then.The formation crystal spends the night in the cold house.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM Tris, pH 8.0 washing crystals 3 times, be suspended in 100mM Tris pH 8.0 then again.At 4 ℃ of preservation crystal.Crystal yield is 60%.

(3) Use the high salt concentration dissolved particles, concentrate subsequently and dialyse, carry out crystallization

In room temperature, the frozen particle (510g) of embodiment 1 is dissolved among 3L 100mM Tris, 1mML-cysteine HCL, 0.5M NaCl, the pH 8.0 2 hours.7000rpm rotary sample 30 minutes, reclaim supernatant.By grossflow filtration (10kD Pall), with sample concentration to 500ml 1 hour, dialysis in 100mM Tris pH 7.5 under agitation then.The formation crystal spends the night in the cold house.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM Tris, pH 7.5 washing crystals 3 times, be suspended in 100mM Tris pH 7.5 then again.At 4 ℃ of preservation crystal.Crystal yield is 67%.

(4) Use the high salt concentration dissolved particles, concentrate subsequently and dialyse, carry out crystallization

In room temperature, the frozen particle (510g) of embodiment 1 is dissolved among 3L 100mM Tris, 1mML-cysteine HCL, 0.5M NaCl, the pH 8.0 2 hours.7000rpm rotary sample 30 minutes, reclaim supernatant.By grossflow filtration (10kD Pall), with sample concentration to 500ml 1 hour, dialysis in 100mM Tris pH 7.0 under agitation then.The formation crystal spends the night in the cold house.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM Tris, pH 7.0 washing crystals 3 times, be suspended in 100mM Tris pH 7.0 then again.At 4 ℃ of preservation crystal.Crystal yield is about 80%.

(5) Use the high salt concentration dissolved particles, concentrate subsequently and dialyse, carry out crystallization

In room temperature, the frozen particle (510g) of embodiment 1 is dissolved among 3L 100mM Tris, 1mML-cysteine HCL, 0.5M NaCl, the pH 8.0 2 hours.7000rpm rotary sample 30 minutes, reclaim supernatant.By grossflow filtration (10kD Pall), with sample concentration to 500ml 1 hour, dialysis in 100mM sodium citrate buffer solution pH 6.5 under agitation then.The formation crystal spends the night in the cold house.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM sodium citrate buffer solution pH 6.5 washing crystals 3 times, be suspended in 100mM sodium citrate buffer solution pH 6.5 then again.At 4 ℃ of preservation crystal.Crystal yield is about 70%.

(6) Use the high salt concentration dissolved particles, concentrate subsequently and dialyse, carry out crystallization

In room temperature, the frozen particle (510g) of embodiment 1 is dissolved among 3L 100mM Tris, 1mML-cysteine HCL, 0.5M NaCl, the pH 8.0 2 hours.7000rpm rotary sample 30 minutes, reclaim supernatant.By grossflow filtration (10kD Pall), with sample concentration to 500ml 1 hour, dialysis in 100mM sodium citrate buffer solution pH 6.0 under agitation then.The formation crystal spends the night in the cold house.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM sodium citrate buffer solution pH 6.0 washing crystals 3 times, be suspended in 100mM sodium citrate buffer solution pH 6.0 then again.At 4 ℃ of preservation crystal.Crystal yield is about 60%.

(7) After homogenization and the dissolving, the OXDC that crystallization is stuck with paste from cell

With the ratio of 1kg cell paste/3L buffer suspension cell again, described buffer contains 100mM Tris pH 7.5,500mM NaCl, 5mM cysteine and 1mM manganese chloride.Spend the night at 4 ℃ of mixing (50-60rpm) cell suspending liquid.Make cell suspending liquid be passed in the homogenizer 2 times of pre-cooling on ice.In test under microscope lysis efficient, with not broken cell suspending liquid with comparing.Suspension is complemented to 10L, mixed 3 hours with outstanding top agitator in room temperature.Then 4 ℃, 7, the centrifugal crude extract 30min of 000rpm reclaims supernatant.Preserve supernatant and granule, be used for SDS-PAGE and characterize.By SDS-PAGE, analyze the expression of OXDC.In supernatant, find most OXDC.Final volume is 10L, and the protein concentration of measurement is 34mg/ml.By grossflow filtration (10kD Pall), sample concentration to 3.5L 1 hour, is under agitation used crystallization buffer (100mM Tris, 100mM NaCl pH 7.5) dilution 1 hour in room temperature then.By centrifugal results crystal, the preservation supernatant is used for analyzing.With 100mM Tris, 100mM NaCl pH 7.5 washing crystals 3 times, be suspended in 100mM Tris and 100mMNaCl, pH 7.5 then again.At 4 ℃ of preservation crystal.

Embodiment 6. crystallizations are from the OXDC of soluble protein.

In shaking bottle, at expression in escherichia coli OXDC.Use 25mM Tris-HCl pH of buffer 8.0, contain the 100mM NaCl of 25U/ml DNA enzyme I, with Micro Fluid instrument (microfluidizer) cell lysis.Room temperature incubation cell lysate 1 hour, make OXDC crystal formation.Rotating crystal is rebuild in 100mM Tris, 100mM NaCl pH 8.

Embodiment 7. is by diffusion of vapor crystallization OXDC.

Use commercial available sparse matrix crystallization test kit: Crystal Screen (Hampton Research; Aliso Viejo, CA), Crystal Screen 2 (HamptonResearch), Wizard I (Emerald Biosystems; Bainbridge Island, WA), Wizard II (Emerald Biosystems), Cryo I (Emerald Biosystems) and Cryo II (Emerald Biosystems) carry out the hanging drop crystallization trial.

600 μ l reagent are placed each hole.3 μ l reagent are dripped on the microscope glass coverslip, 3 μ l OXDC are dripped in the reagent droplet, mix gently.From this 6 μ l reagent and OXDC drop, prepare other 5 drops.Along with the mixing gently of drop, each follow-up (littler) drop has different and unknown albumen/reagent ratio, obtains crystalline probability in the short time period thereby be increased in.After room temperature is incubated overnight, at the crystal of test under microscope hanging drop.Obtain the mass crystallization condition, as shown in table 1.

Table 1: the crystallization condition of OXDC in the hanging drop. ^a

Precipitant	Crystalline description
			40% (v/v) ethanol, 0.1M phosphate-citrate salts, pH 4.20,5% (w/v) PEG 1000	Needle-like
20％(w/v)PEG-3000，0.1M HEPES，pH 7.50，0.2M NaCl	Bar-shaped
		20% (w/v) PEG-3000,0.1M acetate pH 4.5	Bar-shaped, tabular
20% (w/v) PEG 8000, the 0.1M phosphate-citrate salts, pH 4.20,0.2M NaCl	Tabular
		10% (w/v) PEG 8000,0.1M Cacodylate, pH 6.50,0.2 M magnesium acetates	Bar-shaped
9% (w/v) PEG 8000,0.1M Cacodylate, pH 6.50,0.2 M calcium acetates	Needle-like
		40% (v/v) PEG 400, the 0.1M sodium phosphate, pH 6.20,0.2M NaCl	Needle-like
10% (v/v) PEG 8000, the 0.1M sodium phosphate, pH 6.20,0.2M NaCl	Bar-shaped
		20％(w/v)PEG 2000MME，0.1M Tris，pH 7.0	Tabular

^aRecording OXDC concentration by the Bradford test is about 1.7mg/mL.

Embodiment 8. is by little crowd of (microbatch) crystallization OXDC.

By little batch of method, from many crystallization condition crystallization oxalate decarboxylases:

(i) making concentration is that the OXDC of the 10 μ l purification of 23.46mg/ml mixes mutually with 10 μ l 16%PEG8000.2-5 is crystallization immediately in second.The megacryst that forms contains some precipitations.

(ii) making concentration is that the OXDC of the 10 μ l purification of 23.46mg/ml mixes mutually with 10 μ l 20%PEG8000.2-5 is crystallization immediately in second.The megacryst that forms does not contain precipitation.

(iii) making concentration is that the OXDC of the 10 μ l purification of 23.46mg/ml mixes mutually with 10 μ l 24%PEG8000.2-5 is crystallization immediately in second.Form the crystal of littler cube shaped.

(iv) making concentration is that the OXDC of the 10 μ l purification of 23.46mg/ml mixes mutually with 10 μ l 28%PEG8000.2-5 is crystallization immediately in second.Form the crystal of very little cube shaped.Do not precipitate.

(v) making concentration is that the OXDC of the 8 μ l purification of 23.46mg/ml mixes mutually with 12 μ l 24%PEG8000.2-5 is crystallization immediately in second.Form the crystal of very little cube shaped.Do not precipitate.

(vi) making concentration is that the OXDC of the 9 μ l purification of 23.46mg/ml mixes mutually with 11 μ l 24%PEG8000.2-5 is crystallization immediately in second.Form the crystal of small cubes shape.Do not precipitate.

(vii) making concentration is that the oxalate decarboxylase of the 10 μ l purification of 23.46mg/ml mixes mutually with 10 μ l24%PEG 8000.2-5 is crystallization immediately in second.Form the crystal of small cubes shape.Do not precipitate.

(viii) making concentration is that the oxalate decarboxylase of the 11 μ l purification of 23.46mg/ml mixes mutually with 9 μ l24%PEG 8000.2-5 is crystallization immediately in second.Form the crystal of cube shaped.Do not precipitate.

(ix) making concentration is that the OXDC of the 12 μ l purification of 23.46mg/ml mixes mutually with 8 μ l 24%PEG8000.2-5 is crystallization immediately in second.Form the crystal of cube shaped.Do not precipitate.

Embodiment 9. soluble OXDC and the crystalline activity of OXDC

After the dissolved particles, as the soluble OXDC of collection as described in the embodiment 5, rotation, and reclaim supernatant.Behind results and the washing crystal, as described in embodiment 5, collect the OXDC crystal.According to embodiment 15, measure the crystalline activity of soluble OXDC and OXDC.In an experiment, the activity of soluble OXDC is 12 units/mg, and the crystalline activity of OXDC is 35 units/mg.

As the soluble OXDC of collection as described in any among the embodiment 2-5, and as results OXDC crystal as described in any among the embodiment 2-8.According to embodiment 15, measure soluble and activity crystal OXDC.The crystalline activity of OXDC can be the active at least about 100%, 200%, 300%, 400% or 500% of soluble OXDC.

Embodiment 10. uses glutaraldehyde cross-linking oxalate decarboxylase crystal.

Use the oxalate decarboxylase crystal of glutaraldehyde cross-linking according to any preparation among the embodiment 2-8.After the crystallization, the OXDC crystal is concentrated into 20-30mg/ml.0.8ml 25% glutaraldehyde is added the 20ml crystal, make 1% glutaraldehyde solution, at roll crystal 18 hours of room temperature.With 100mM Tris, the crosslinked crystal 5 of pH 7.00 washings, be suspended in again among 10mM Tris, the pH 7.00.

The ratio of contrast lenticular OXDC and crosslinked OXDC (being called OXDC-CLEC) is lived (6 tests), shows in different goods, and what crosslinked oxalate decarboxylase crystal kept the proteic original activity of lenticular surpasses 30% to surpassing 50%.

For the influence of the glutaraldehyde of testing variable concentrations, at pH 8.0, at 25 ℃, with glutaraldehyde (from 0.05% to 2%, final concentration) the crystalline 1ml aliquot of crosslinked OXDC (60mg/ml) of variable concentrations 18 hours to enzymatic activity.By in Eppendorf tube, separating crosslinked crystal in that 2000rpm is centrifugal, thereby stop crosslinkedly, then crosslinked crystal is suspended in again among 100mM TrisHCl, the pH 7.0 of 1ml.Wash crosslinked OXDC (OXDC-CLEC) 5 times with 100mM TrisHCl pH of buffer 7.5 then, subsequently with 3 times (referring to the result of following table 2) of 10mM TrisHCl pH of buffer 7.5 washings.

The dissolubility that the embodiment 11. crosslinked crystalline pH of oxalate decarboxylase control.

Make pH be reduced to 3.0, the different crosslinked crystalline dissolubility of oxalate decarboxylase of inspection from 7.5.At 1mg/ml, the crosslinked crystal of incubation in 50mM glycine HCl (pH 3.0).Under agitation, after 5 hours, take out aliquot at 37 ℃ of incubations.After the not molten crosslinked crystal of 2000rpm centrifugalize also passes through 0.22 μ m filter filtering supernatant, at OD ₂₈₀Nm measures soluble protein concentration.The result is as described in the following table 2.

Table 2: control with the glutaraldehyde cross-linking OXDC crystal of different weight percentage and the pH of OXDC-CLEC The dissolubility of system

Sample	The % glutaraldehyde	The leaching of % albumen
			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 show, in the presence of having at least about 0.05% (final concentration) glutaraldehyde, form the stable basically crosslinked crystal of glutaraldehyde OXDC.

The pH living features of embodiment 12. soluble OXDC, lenticular OXDC and OXDC-CLEC

By adding glutaraldehyde (Sigma), crosslinked oxalate decarboxylase crystal as preparation as described in the embodiment 2-8.At 25 ℃, 1% glutaraldehyde (final concentration) the crystalline 1ml aliquot of crosslinked OXDC (30-40mg/ml) of usefulness pH 8.0 18 hours.By in Eppendorf tube, separating crosslinked crystal in that 2000rpm is centrifugal, thereby stop crosslinkedly, crosslinked crystal is suspended in again among 1ml 100mM Tris HCl, the pH 7.0 then.Wash OXDC-CLEC 5 times with 100mM TrisHCl pH of buffer 7.0 then, subsequently with 10mM TrisHCl pH of buffer 7.0 washings 3 times.Use different buffer and pH,, measure the pH living features of OXDC-CLEC by the crystalline activity of measurement as described in embodiment 15: 50mM glycine HCl buffer, in pH 2.0 and 3.0; 50mM succinate buffer is in pH 4.0,5.0 and 6.0; With 50mM Tris buffer, at pH 7.0.Be determined at the activity level 2 times of each pH, calculate average activity.Result shown in Figure 1 shows, OXDC-CLEC activity of solvable homologue than it between pH 3.5 to 6.0 is higher.Uncrosslinked crystal shows the activity more much higher than the soluble form of oxalate decarboxylase at different pH, and from about 50% to about 200%, 300% or 400% is higher.

Oxalate decarboxylase treatment in the embodiment 13. intestinal originality oxaluria animal models.

The Hyperoxaluric rat model of intestinal originality, dosage range research:Male Sprague Dawley (SD) rat of high oxalates diet fed constitutes and to be applicable to the Hyperoxaluric animal system of research intestinal originality.In this research, use 1.1% diet potassium oxalate and cause urinary oxalate to increase by 5 to 10 times.

Is that Sprague Dawley (SD) rat that 100-120 restrains is divided into matched group and experimental group (every group of 5 rats) at random with 20 less than 35 day age and weight.Before treatment, make rat adaptation metabolic cage (LabProducts, Inc. separately; Seaford, DE) 7 days.In this stage, unrestrictedly supply with additional acidifying water for rat, and feed and contain synthetic diet (the Research Diets TD89222PWD of 1.1% potassium oxalate and low (0.5%) concentration calcium; Harlan Teklad; Madison, WI).During treating, rat is kept this diet.

After the adaptive phase, will with 1% glutaraldehyde be mixed with crosslinked crystal (referring to, for example, embodiment 9) the reorganization oxalate decarboxylase of 3 various dose be administered to 4 of experimental rats week continuously.Crystal as solidify/exsiccant food enzymatic mixture oral (5,25 and 80mg OXDC-CLEC slurry in 10mM TrisHCl pH 7.0, mix with 15g food independently of one another, and sets/dries; In each morning, load about 20g food/enzymatic mixture again for food container).Before treatment, the basic urinary oxalate based on them is divided into matched group and experimental group at random with rat.

The analysis of urine sample:Collect the twenty-four-hour urine sample in the metabolic cage on acid (250 μ l 6N hydrochloric acid are mixed mutually with the urine sample of collecting) in 24h, so that the urine ascorbic acid is minimized to the spontaneous decomposition of oxalates.Before further analyzing, at-70 ℃ of preservation samples.Collect the urine of every day and a plurality of 24h (hour) urine sample, be used for oxalates and kreatinin and measure.The mensuration of oxalates and kreatinin is as described in the embodiment 15.The homaluria of oxalates and kreatinin is expressed as the μ mol of detected oxalates and kreatinin in the 24h urine sample.Use Student t-check, all data of statistical analysis.

As shown in Figure 2, give the oral OXDC-CLEC of chronic Hyperoxaluric SD rat, cause from treating the lasting reduction of the 4th day beginning urinary oxalate.Noting the maximum of 25-40% in maximum dose level group (80mg OXDC-CLEC) reduces continuously.More the 5mg of low dosage and 25mg OXDC-CLEC cause the littler reduction (in the 25mg group up to 30%, in the 5mg group up to 20%) of urinary oxalate.The dosage of 25mg and 80mg produces significantly in all test days (except 25mg group the 21st beyond the highest heavens) and reduces, and the while, lowest dose level 5mg had inapparent smallest effect.This result has disclosed the dose dependent effect of OXDC-CLEC treatment.

Embodiment 14: the oral oxalate decarboxylase treatment in the primary hyperoxaluria animal model.

The mouse model of I type primary hyperoxaluria:The mice that AGT1 knocks out lacks liver peroxidase alanine: the glyoxalic acid aminotransferase, promptly cause the defective of I type primary hyperoxaluria.In C57B16 and the strain of 129/sv background, gomphosis mouse bred to become to isozygoty and be.(0.2mmol/L) compares with wild type, and all Agxt mices of isozygotying show slight oxaluria (1-2mmol/L), and urinary oxalate than normal value rising 5-10 doubly.Also find 30-50% male and 0% the female calcium oxalate stone that develops into slight nephrocalcinosis and urethra in later stage of life (4-7 monthly age).Interesting ground, when analyzing sudden change in C57B16 strain of the same race, the oxaluria in any sex is uncorrelated with the urinary stone development; This has emphasized frequent observed phenotypic variability in this disease.

In these experiments, use totally 44 male mices (strain AGT1KO/129sv, developer are Dr.Salido, La Laguna Tenerife, Spain).Mice is divided into matched group and 3 experimental grouies at random.Mice weight is the 20-25 gram, less than 6 monthly ages.

Spent glycol (EG) is attacked AGT1KO (129sv) mice, to cause serious oxaluria and form the calcium oxalate deposit in excess of the kidney matter.EG is a kind of common alcohol that is metabolized to oxalates in liver.Usually, attack 2-6 after week at EG, the AGT1KO mice in the 129/sv background shows impaired renal function sign, and this can followingly measure: (i) drainage of the variation of urine medium-height grass hydrochlorate; The (ii) creatinine clearance of Jiang Diing and (iii) finally cause renal failure and dead nephrocalcinosis.

Before treatment, make mice adapt to each metabolic cage (Tecniplast USA Inc, Exton, PA USA) 7 days, feeds and contains standard breeding stock diet (17% albumen less than 0.02-0.08% oxalates and about 0.5-0.9% calcium, 11% fat, 53.5% carbohydrate).After the adaptive phase, mice is divided into 4 groups; 3 treatment groups are fed and the blended oxalate decarboxylase-CLEC of food, and the mice in the matched group of coupling accepts not add the identical diet of experiment thing.From treating first day, the drinking water that has added 0.7%EG unrestrictedly is provided for all mices to the research end.Attack after several days, mice drains about 3-6mmol/L oxalates every day in their urine, and this than the high about 10-20 of wild type (not attacking) mice doubly.

Using of OXDC-CLEC enzyme; Dosage range research:In the dose study of OXDC-CLEC, use totally 44 male mices from the AGT1KO/129sv strain.Mice weight is the 20-25 gram, less than 6 monthly ages.Attack mice with EG, be divided into matched group and experimental group then at random.Supervision is mixed with crosslinked crystal (1% glutaraldehyde; Referring to embodiment 9) 4 of effects week continuously of reorganization oxalate decarboxylase of 3 various dose.The term of Shi Yonging " OXDC-CLEC " is meant as being mixed with the reorganization oxalate decarboxylase of crosslinked crystal (1% glutaraldehyde) as described in the embodiment 9 in this embodiment.5,25 and 80mg/ days nominal standard dose, OXDC-CLEC as solidify/exsiccant food enzymatic mixture is oral.The enzyme slurry in 10mM Tris-HCl buffer (pH 7.0) of q.s is mixed mutually with 3.5g food, and sets/dries.In each morning, load about 7g food/enzymatic mixture again for food container.

The assessment of the effect of OXDC-CLEC:By urinary oxalate reduction, sedimentary prevention and the survival rate of calcium oxalate in excess of the kidney matter, monitor the effect of enzyme treatment.When research finishes, put to death the mice of survival, blood sampling is used for kreatinin and measures.

The analysis of urine sample:Collect the twenty-four-hour urine sample in the metabolic cage on acid (50 μ l 6N hydrochloric acid/3-4ml urine), so that the urine ascorbic acid is minimized to the spontaneous decomposition of oxalates.Before further analyzing ,-20 ℃ of preservation urine samples.Collect urine and a plurality of 24h urine sample of every day, analyze oxalates and creatinine levels.The mensuration of oxalates and kreatinin is as described in the embodiment 15.The homaluria of oxalates and kreatinin is expressed as the oxalates that is excreted in the 24h urine sample (mL) and the μ mol of kreatinin.Use Student t-check, statistical data analysis.

The analysis of blood sample:When research finishes, put to death mice, collect blood serum sample.Measure for serum creatinine, the Jaffe reaction method of use slight improvement (referring to, for example from Oxford Medical Research, the dull and stereotyped test kit of the kreatinin microdetermination of Inc.; Slot, Scand J.Clin.Lab.Invest. 17: 381,1965; With Heinegard D, Clin.Chim.Acta 43:305,1973).In test tube, the undiluted blood serum sample of 80 μ l is mixed mutually with 800 μ l picrins, room temperature incubation 30 minutes.Measure colour developing at the 510nm spectrophotometer method; Add 33.3 μ l, 60% acetic acid then, with the cancellation nonspecific reaction.Thorough biased sample is at the room temperature incubation after 5 minutes, once more at the 510nm reading.Final absorbance is expressed as the poor of 2 readings.The serial dilutions of 1mM kreatinin solution is used for standard curve.

By measuring creatinine clearance, measure renal function indirectly.Creatinine clearance is expressed as the discharge rate (U of kreatinin _CrX V) (U wherein _CrRepresent the kreatinin concentration (μ mol/L) in the urine sample) divided by plasma creatinine (P _Cr).This is expressed as:

C _cr＝(U _crx V)/P _cr＝mL/h

The security parameter that monitors in research process is that mortality rate, food and water are taken in and body weight.In research process, carry out mortality rate inspection and the objective observation of cage side every day once.Measure food intake every day, write down water weekly and take in.When beginning research and research finish, write down the body weight of all animals.

As shown in Figure 3, to the oral OXDC-CLEC of AGT1KO (129sv) mice that EG attacks, cause comparing the remarkable reduction of urinary oxalate level with the untreated control mice of coupling from treating when finishing to research in the 4th day.In all 3 treatment groups, observe 30 to 50% reduction, in maximum dose level group (80mg OXDC-CLEC), observe maximum the reduction.The urinary oxalate that the more low dosage of 25mg and 5mgOXDC-CLEC produces up to 35% reduces.

By azygous pair of tail Student t-check, analysis result.When the research beginning, each administration group has the n=11 mice, attacks but several mices die from ethylene glycol in research process.Shown result only is included in the mice of the certain day survival of urinary oxalate measurement.The initial rising of observing urinary oxalate causes the kidney filtering function of nephrocalcinosis, reduction and oxalates discharge rate reduction and the final down death of worst condition in time subsequently, and they can explain the excretory bell sigmoid curves of urinary oxalate in the matched group best.

Measure the assessment renal function by creatinine clearance.When research finishes, put to death and live through all animals that EG attacked for 4 weeks, collect blood and measure plasma creatinine and creatinine clearance.11 mices of all of 80mg dosage group live through EG and attacked for 4 weeks; 8/11 mice survival of 25mg OXCD-CLEC dosage group; 8/11 mice survival of 5mg OXCD-CLEC dosage group; 7/11 mice survival of matched group.Measure for serum creatinine, use above-mentioned slight improvement the Jaffereaction method (referring to, for example from Oxford Medical Research, the dull and stereotyped test kit of the kreatinin microdetermination of Inc.; Slot, Scand J.Clin.Lab.Invest. 17: 381,1965; With Heinegard D, Clin.Chim.Acta 43:305,1973).

By measuring creatinine clearance, assess the effect of oral OXDC-CLEC to renal function.Live through whole month conceptual phase mice creatinine clearance as shown in Figure 4.When contrasting, accept the creatinine clearance significantly higher (p＜0.05) of the survival mice of 80mg OXDC-CLEC with matched group.

All mices (11/11) of 80mg OXDC-CLEC treatment group live through 4 all EG attack options, and in the matched group only 7 mices (7/11) live through this scheme.The kidney filtering rate of measuring by creatinine clearance also significantly is lower than 80mg dosage group (Fig. 4).

The nephridial tissue pathological analysis:Paraffin embedding is handled little Ren Mus routinely, and the location, so that obtain the complete transverse section of kidney.With each 4 μ m that cut into slices, each kidney is cut into 12 serial section, with hematoxylin and eosin dyeing, be used for conventional organization and learn inspection, or detect the existence of calcium oxalate crystals in nephridial tissue by specificity Yasue metal replacement histochemical method.Use the 20X amplification, at the test under microscope microscope slide, inspection personnel's section of marking under 4-class scale is applied to identical standard each anatomic region (cortex, medullary substance and mastoid process) of kidney.Scoring is that (i) do not have (not having the oxalates crystal in any zone); (ii) minimum (1-5 crystal being arranged) at arbitrary region; (iii) medium (6-10 crystal being arranged) at arbitrary region; (iv) serious (All Ranges has a plurality of crystal set).

From the presentation graphics of treatment and the nephridial tissue of control animal shown in Fig. 5 A-5C.At the 20x amplification, the visible male calcium oxalate crystals of Yasue-in excess of the kidney matter.Organize all mices with the treatment of 80mg OXDC-CLEC treatment and have the normal healthy kidney, do not have calcium oxalate deposit vestige (Fig. 5 A).In matched group and in some mice, observe medium nephrocalcinosis (Fig. 5 B) and serious nephrocalcinosis (Fig. 5 C) from the low dose therapy group.White arrow indication calcium oxalate deposit among Fig. 5 C, the grey arrow indication has the big zone of interstitial fibrosis.

Use specificity Yasue metal replacement method that the histological examination of kidney is disclosed, deposit mainly is present in the cortex and the medullary substance part of kidney.Under the situation of serious nephrocalcinosis (Fig. 5 C), calcium oxalate deposit random distribution in kidney.Also observe the sign of fibrosis and inflammation, the form of glomerule changes, and the calcium oxalate deposit forms in glomerule once in a while.Behind necropsy, have normal kidney form from all mices (11/11) of 80mg dosage group, there is not calcium oxalate sedimentary vestige in kidney or bladder.On the contrary, 100% (11/11) mice from untreated matched group has the calcium oxalate deposit.At low treatment group (25mg or 5mg OXDC-CLEC), 63% (7/11) mice has the calcium oxalate deposit in kidney.These results confirm that the OXDC-CLEC oral medication is to the effect positive, dose dependent of the prevention of Hyperoxaluric reduction and kidney mesoxalic acid calcium crystal deposition in the AGT1KO mice that EG attacks.Summary from the histopathological analysis of all 4 groups of mices is as shown in table 3.

Treatment group and contrast behind the seriousness of table 3. nephrocalcinosis and the OXDC-CLEC oral medication Get involved in the group number of mice

Also estimated the OXDC-CLEC oral medication forms frequency to control mice and the urinary stone from the mice of 3 different treatment groups effect.2 big class calcium in bladder, have been found: calcium oxalate monohydrate calculus and calcium oxalate dihydrate calculus from EG AGT1KO mice.In 36% (4/11) mice of matched group and in 19% (2/11) mice of 2 lower treatment groups, there is obvious visible vesical calculus.In 80mg high dose OXDC-CLEC group, do not observe vesical calculus.X-ray diffraction the analysis showed that the calculus with albescent, coarse bud shape surface mainly is made up of the calcium oxalate monohydrate, and the relative bigger calculus correspondence with sharp keen crystal angle the calcium oxalate dihydrate.

Carry out survival rate analysis by the Kaplan-Meier estimator.Use the Kaplan-Meier method, wherein will be in the experimenter's of some time point death survival rate divided by under study for action at this number of the experimenter of survival still constantly, analyze the influence of the survival rate of the mice that the OXDC-CLEC treatment attacks ethylene glycol.The difference (Fig. 6) between the group in the research that this method has been used graphic rendition.Statistical procedure for example Kaleida figure and STATS through being usually used in calculating.

Compare with the contrast of coupling, the OXDC-CLEC oral medication can increase the survival rate of the AGT1KO mice of EG attack.All mices (11/11) from 80mg OXDC-CLEC treatment group live through 30 days conceptual phases, and do not have ill sign, and have serious nephrocalcinosis from 4/11 mice of matched group, and the development urinary stone.

Because the OXDC-CLEC intention is used for oral administration, has estimated the probability of crosslinked crystal to the detrimental effect of gastrointestinal (GI) tissue.The digestive tract of the AGT1KO mice that the EG of macroscopy treatment attacks carries out histologic analysis with hematoxylin-eosin dyeing to the gastrointestinal tract different piece that comprises stomach (substance and hole) and small intestinal (jejunum and ileum).Estimate and confirm that 4 weeks of OXDC-CLEC oral medication are well-tolerated, and can not cause gastrointestinal structure or metamorphosis.Observe similar result for large intestine.

The dosage range of the AGT1KO mice of attacking with the EG of oxalate decarboxylase-CLEC oral medication The summary of researchOXDC-CLEC oral medication in the primary hyperoxaluria animal model is safe and efficient.In a word, make urinary oxalate reduce 30-50% in 4 weeks of OXDC-CLEC oral medication.Use the experiment thing of the evaluation of 3 dosage, write down significant and lasting reduction.Calcium oxalate deposition in the highest therapeutic dose oral medication 4 weekly assemblies prevention excess of the kidney matter.Treat 4 weekly assemblies at 2 lower oral doses and improve survival rate, can prevent the animal dead rate at the highest research dosage.At last, 4 all therapeutic schemes do not produce the variation of macroscopic view or microcosmic in gastrointestinal tract.

Embodiment 15. measures

Determination of protein concentration:By measuring absorbance, measure the concentration of oxalate decarboxylase at 280nm.1.36 the absorbance of optical density (OD) is regarded 1mg/ml as.

The OXDC determination of activity:Use improved Sigma Aldrich scheme (enzyme assay of oxalate decarboxylase EC4.1.1.2), measure the activity of soluble oxalate decarboxylase, oxalate decarboxylase crystal and crosslinked oxalate decarboxylase crystal (OXDC-CLEC).This is a kind of two indirect step determinations of activity; In first reaction, OXDC changes into formates and carbon dioxide with the substrate oxalates.In second reaction, formates hydrogen is transferred to NAD by formates dehydrogenase stochiometrically, forms NADH.The follow-up concentration of quantitative NADH on 340nm spectrophotometer ground.As the unit of the enzymatic activity of giving a definition (u): the oxalate decarboxylase of a unit forms formates and carbon dioxide at pH 5 and 37 ℃ of per minutes from 1.0 μ mol oxalates.

For OXDC crystal and OXDC-CLEC respectively in the concentration of 0.007-0.02 and 0.009-0.03mg/mL, standardization working sample in containing the 5mM kaliumphosphate buffer pH 7.0 of 1mM DTT (Sigma).By absorbance, measure protein concentration at 280nm.Before use at cold diH ₂The formates dehydrogenase (FDH) of preparation 40U/mL among the O, and place on ice.All other reagent remain on room temperature.In the following manner, with stirring rod reagent is added the 2ml micro test tube: mix 300 μ l 100mM potassium phosphates and 200 μ l potassium oxalate pH 4.0, at 37 ℃ of warm 5min in water-bath, the 5mM potassium phosphate that then 100 μ l is contained 1mM DTT adds in the blank bottle, and the oxalate decarboxylase that 100 μ l are diluted adds in all other bottles.After 2 minutes, with 150mM dipotassium hydrogen phosphate cessation reaction.In second reaction, 25 μ l NAD solution are added 100 μ l FDH solution, continue the other 20min of incubation.Then 16, the centrifugal all samples 1min of 100rpm.Then reactant mixture is transferred to 1.5mL ultraviolet test glass, (Shimadzu Scientific Instruments, Columbia Maryland), are determined at the absorbance of 340nm, and record to use Shimadzu BioSpec.

Following calculating enzyme is than living:

Unit/mL OXDC= [absorbance x cumulative volume (1.725mL) x dilution gfactor]

(Ext.NADH=6.22) (Vol.OXDC=0.1mL) (minute=2/5min)

In a concrete experiment, with crosslinked oxalate decarboxylase crystal (OXDC-CLEC) (by spend the night rhomboidan with 1% glutaraldehyde cross-linking of upset; Referring to embodiment 9) compare with the OXDC crystal.Active 50% of the lenticular OXDC goods that the OXDC-CLEC maintenance is corresponding.

Measure oxalates by the colourity method:The oxalates colourity test kit that is used for quantitative assay urine medium-height grass hydrochlorate available from Trinity Biotech USA (St.Louis, MO) or GreinerDiagnostic AG (Dennliweg 9, Switzerland).According to manufacturer's description, dilution and processing urine sample.Mensuration comprises 2 enzyme reactions: (a) oxalate oxidase is oxidized to carbon dioxide and hydrogen peroxide with oxalates, (b) hydrogen peroxide that forms like this reacts with 3-methyl-2-[4-morpholinodithio quinoline ketone hydrazone (MBTH) and 3-(dimethylamino) benzoic acid (DMAB) having in the presence of the peroxidase, the indamines dyestuff that formation can detect at the 590nm absorbance.The intensity of the color that produces and the concentration of sample medium-height grass hydrochlorate are directly proportional.Calculate the urinary oxalate value from standard curve.

Measure kreatinin by the colourity method:The kreatinin colourity test kit that is used for quantitative assay urine kreatinin is available from Quidel Corporation (San Diego, CA; The METRA kreatinin is measured test kit) or Randox Laboratories (Antrim, United Kingdom).This mensuration is based on following principle, i.e. the reaction of picric acid in kreatinin and the aqueous slkali forms the product that has at the absorbance of 492nm.The amount and the kreatinin concentration of the complex that forms are directly proportional.15 times of the 24h rat urine samples of collecting from single metabolic cage with DDW dilution.The urine sample of 20 μ l dilution is mixed mutually with 20 μ l picric acid/sodium hydroxide (1: 1).After 2 minutes, measure absorbance at the room temperature incubation at 492nm.Calculate urine creatine acid anhydride value from standard curve.

Embodiment 16. is used for the treatment of the OXDC treatment of people's oxalates associated conditions

By oral crosslinked oxalate decarboxylase crystal, can treat needs treatment or for example Hyperoxaluric people of prevention oxalates associated conditions.Approximate dosage at 10 μ g/kg to 25mg/kg, 1mg/kg to 25mg/kg or 5mg/kg to 100mg/kg is used the oxalate decarboxylase crystal, and this is determined by the treatment clinicist, and depends on the seriousness of symptom and the progress of disease.Use the crosslinked crystal of oxalate decarboxylase every day 1,2,3,4 or 5 times, or use with lower frequency, for example weekly or 2 times.Oral like this OXDC-CLEC causes the urinary oxalate level to reduce at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or more.

The recombinant production of embodiment 17. oxalate decarboxylases

In human embryo kidney (HEK293) cell:DNA (for example, SEQ IDNO:1 or the 2) clone of coding OXDC is advanced in the suitable expression vector.Sequence with the carrier linearisation, is used Lipofectamine after confirming in 6cm diameter dish ^TM2000 transfection reagents transform the pre-HEK293 cell of inoculating with linearizing carrier.In appropriate culture medium, cultivate the transfection reactant and spend the night, in the culture medium of having added the 0.5g/L neomycin, select transformant then.Growth is differentiated the HEK293 cell clone of stable transfection up to after 3 weeks in containing the culture medium of neomycin.Separate then and breed the clone, and be used for OXDC and express.

In Chinese hamster ovary (CHO) cell:The dna clone of coding OXDC gene is advanced in the suitable expression vector.By trypsinization, make the CHO lec 3.2.8.1 cell separation of cultivation then, gather in the crops by centrifugal.Then with cell suspension in the brine buffer solution (EPBS) of electroporation phosphate-buffered to～1 * 10 ⁷The final concentration of/ml transforms with linearizing carrier by electroporation.After the incubated overnight, culture medium is replaced by the culture medium of having added the 0.5g/L neomycin.Carry out the continuous replacing of culture medium, with the Chinese hamster ovary celI clone of screening stable transfection.In case set up and breed the cell clone of stable transfection, cell is used for OXDC expresses.

In pichia pastoris:The dna clone of coding OXDC gene is advanced in the suitable expression vector.After sequence is confirmed, with the carrier linearisation, transform then the pichia pastoris host cell (referring to, people such as Whittaker, J.Biol.Inorg.Chem. 7: 136-145,2002).Select transformant with Zeocin, amplification in buffered glycerol complex medium (BMGY), and use methanol induction.Can separate OXDC from culture medium then.

In saccharomyces cerevisiae:Synthetic OXDC gene clone can be advanced suitable expression vector, the terminator sequence that the latter is contained Gall promoter (pGal) for example and expressed.Sequence transforms into competence saccharomyces cerevisiae W303-1A by electroporation with expression vector after confirming.Screening transformant, and breeding are used for OXDC then and express.

In insect cell:The dna clone of coding OXDC can be advanced suitable expression vector, for example, rhabdovirus system.Sequence transforms into competence DH10Bac Bacillus coli cells with carrier after confirming.Screening and confirmation contain the Bacillus coli cells of the rod granule of recombinating.The bacmid dna that separates reorganization, and use for example Cellfectin of reagent ^TM(Invitrogen, Carlsbad California), are used for transfection insecticide Sf9 cell to reagent.Can separate the recombinant baculovirus granule then, breeding, and titration are used to infect the Sf9 cell then, carry out OXDC and express.

In escherichia coli:The dna clone of coding OXDC can be advanced suitable coli expression carrier.Sequence transforms into competence e. coli bl21 or escherichia coli Origami B (DE3) where necessary with carrier after confirming, forms disulfide bond in the recombiant protein that the latter allows to express in this bacterial strain.By cultivating transformant containing on the antibiotic nutrition flat board, the screening transformant, and use the primer of OXDC gene specific, PCR confirms by bacterium colony.Cultivate transformant in the liquid medium within then, and induce, be used for OXDC and express with isopropyl-(IPTG).

Embodiment 18. sequences

The JINZHENGU sequence that will in Candida boidinii (Candida boidinii), express (SEQ ID NO:1)2 NotI sequences indicate underscore; The ATA triplet of runic is the interval codon; The sequence that indicates double underline is the α mating factor sequence of optimizing for Candida boidinii; The OXDC coded sequence is represented with lower case.

1ataagaat GCGGCCGCATA

50

100

150

200

250 atgtttaataatt

300ttcaaagattattaactgttattttattatctggttttactgctggtgtt

350ccattagcttctactactactggtactggtactgctactggtacttctac

400tgctgctgaaccatctgctactgttccatttgcttctactgatccaaatc

450cagttttatggaatgaaacttctgatccagctttagttaaaccagaaaga

500aatcaattaggtgctactattcaaggtccagataatttaccaattgattt

550acaaaatccagatttattagctccaccaactactgatcatggttttgttg

600gtaatgctaaatggccattttctttttctaaacaaagattacaaactggt

650ggttgggctagacaacaaaatgaagttgttttaccattagctactaattt

700agcttgtactaatatgagattagaagctggtgctattagagaattacatt

750ggcataaaaatgctgaatgggcttatgttttaaaagggtctactcaaatt

800tctgctgttgataatgaagggagaaattatatttctactgttggtccagg

850tgatttatggtattttccaccaggtattccacattctttacaagctactg

900ctgatgatccagaaggttctgaatttattttagtttttgattctggtgct

950tttaatgatgatggtacttttttattaactgattggttatctcatgttcc

1000aatggaagttattttaaaaaattttagagctaaaaatccagctgcttggt

1050ctcatattccagctcaacaattatatatttttccatctgaaccaccagct

1100gataatcaaccagatccagtttctccacaagggactgttccattaccata

1150ttcttttaatttttcttctgttgaaccaactcaatattctggtgggactg

1200ctaaaattgctgattctactacttttaatatttctgttgctattgctgtt

1250gctgaagttactgttgaaccaggtgctttaagagaattacattggcatcc

1300aactgaagatgaatggactttttttatttctggtaatgctagagttacta

1350tttttgctgctcaatctgttgcttctacttttgattatcaaggtggtgat

1400attgcttatgttccagcttctatgggtcattatgttgaaaatattggtaa

1450tactactttaacttatttagaagtttttaatactgatagatttgctgatg

1500tttctttatctcaatggttagctttaactccaccatctgttgttcaagct

1550catttaaatttagatgatgaaactttagctgaattaaaacaatttgctac

1600taaagctactgttgttggtccagttaattaa GCGGCCGCtaaactat 1646

Bacillus subtilis sequence (SEQ ID NO:2): 705 " G " that indicate underscore is meant A → G base substitution in the position.This base substitution can not change aminoacid sequence.

1ATGAAAAAACAAAATGACATTCCGCAGCCAATTAGAGGAGACAAAGGAG

50CAACGGTAAAAATCCCGCGCAATATTGAAAGAGACCGGCAAAACCCTGAT

100ATGCTCGTTCCGCCTGAAACCGATCATGGCACCGTCAGCAATATGAAGTT

150TTCATTCTCTGATACTCATAACCGATTAGAAAAAGGCGGATATGCCCGGG

200AAGTGACAGTACGTGAATTGCCGATTTCAGAAAACCTTGCATCCGTAAAT

250ATGCGGCTGAAGCCAGGCGCGATTCGCGAGCTTCACTGGCATAAAGAAGC

300TGAATGGGCTTATATGATTTACGGAAGTGCAAGAGTCACAATTGTAGATG

350AAAAAGGGCGCAGCTTTATTGACGATGTAGGTGAAGGAGACCTTTGGTAC

400TTCCCGTCAGGCCTGCCGCACTCCATCCAAGCGCTGGAGGAGGGAGCTGA

450GTTCCTGCTCGTGTTTGACGATGGATCATTCTCTGAAAACAGCACGTTCC

500AGCTGACAGATTGGCTGGCCCACACTCCAAAAGAAGTCATTGCTGCGAAC

550TTCGGCGTGACAAAAGAAGAGATTTCCAATTTGCCTGGCAAAGAAAAATA

600TATATTTGAAAACCAACTTCCTGGCAGTTTAAAAGATGATATTGTGGAAG

650GGCCGAATGGCGAAGTGCCTTATCCATTTACTTACCGCCTTCTTGAACAA

700GAGCCGATCGAATCTGAGGGAGGAAAAGTATACATTGCAGATTCGACAAA

750CTTCAAAGTGTCTAAAACCATCGCATCAGCGCTCGTAACAGTAGAACCCG

800GCGCCATGAGAGAACTGCACTGGCACCCGAATACCCACGAATGGCAATAC

850TACATCTCCGGTAAAGCTAGAATGACCGTTTTTGCATCTGACGGCCATGC

900CAGAACGTTTAATTACCAAGCCGGTGATGTCGGATATGTACCATTTGCAA

950TGGGTCATTACGTTGAAAACATCGGGGATGAACCGCTTGTCTTTTTAGAA

1000ATCTTCAAAGACGACCATTATGCTGATGTATCTTTAAACCAATGGCTTGC

1050CATGCTTCCTGAAACATTTGTTCAAGCGCACCTTGACTTGGGCAAAGACT

1100TTACTGATGTGCTTTCAAAAGAAAAGCACCCAGTAGTGAAAAAGAAATGC

1150AGTAAATAA 1158

Oxalate decarboxylase albumen (Swiss-Prot:O34714) as follows (SEQ ID NO:3) from the translation of bacillus subtilis sequence.

1MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMK

50FSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKE

100AEWAYMIYGSARVTIVDEKGRSFIDDVGEGDLWYFPSGLPHSIQALEEGA

150EFLLVFDDGSFSENSTFQLTDWLAHTPKEVIAANFGVTKEEISNLPGKEK

200YIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPIESEGGKVYIADST

250NFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGH

300ARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWL

350AMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK 385

Embodiment 19. soluble OXDC, lenticular OXDC and OXDC-CLEC are when hanging down pH 3.0 Stability

The OXDC-CLEC (embodiment 10) of the lenticular OXDC (embodiment 5) of soluble OXDC (embodiment 5), the 5.0mg/mL of 2.5mg/mL and 5.0mg/mL is placed in each 1mL sodium citrate buffer solution pH 3.0 in the Eppendorf tube, at 37 ℃ of incubation 5h.Sampling in 0,2 and 5 hour, be used to measure the stability of enzyme.As described in embodiment 15, measure activity.Show that OXDC and OXDC-CLEC after 0,2 and 5 hour are in the result of the stability of pH 3.0 as shown in Figure 7.With compare at first, behind pH 3.0 incubation 5h, OXDC-CLEC keeps about 100% activity, and soluble OXDC pro-2h in the forfeiture about 51% activity, behind 5h, only keep about 40% activity.Lenticular OXDC was more stable than soluble OXDC, kept about 68% activity at 1 hour, kept about 67% activity at 2 hours.

Embodiment 20. soluble OXDC, lenticular OXDC and OXDC-CLEC crystal are having the stomach egg Stability under white enzyme exists

The OXDC-CLEC (embodiment 10) of the lenticular OXDC (embodiment 5) of soluble OXDC (embodiment 5), the 10.0mg/mL of 1.0mg/mL and 1.0mg/mL is placed in each 1mL sodium citrate buffer solution pH 3.0 in the Eppendorf tube, at 37 ℃, with OXDC: pepsin is 50: 1 a ratio, (concentration of pepsin stock solution is 1mg/mL with pepsin, in 25mM Tris-HCL buffer, pH 7.5) incubation 5h.Sampling in 0,2 and 5 hour, be used to measure the stability of enzyme.As described in embodiment 15, measure activity.Show that soluble OXDC, lenticular OXDC and OXDC-CLEC are in the result that the stability in the presence of the pepsin is arranged as shown in Figure 8 after 0,2 and 5 hour.

As shown in Figure 8, at low pH with having in the presence of the pepsin, having disclosed the OXDC-CLEC preparation surpasses soluble OXDC.With compare at first, behind 37 ℃ of incubation 5h, OXDC-CLEC keeps about 60% activity, and most of soluble OXDC and uncrosslinked lenticular OXDC are degraded by pepsin behind 2h, only keep about 20% activity behind 5h.

Embodiment 21. soluble OXDC, lenticular OXDC and OXDC-CLEC are having pancreas curdled milk egg Stability under white enzyme exists

The OXDC-CLEC (embodiment 10) of the lenticular OXDC (embodiment 5) of soluble OXDC (embodiment 5), the 10.0mg/mL of 1.0mg/mL and 1.0mg/mL is placed in each 1mL 25mM Tris-HCl pH of buffer 7.5 in the Eppendorf tube, at 37 ℃, with OXDC: chymase is 50: 1 a ratio, (concentration of chymotrypsinogen liquid is 1mg/mL with chymase, in 25mM Tris-HCL buffer, pH 7.5, prepared fresh) incubation 5h.Sampling in 0,2 and 5 hour, be used to measure the stability of enzyme.As described in embodiment 15, measure activity.Show that soluble OXDC, lenticular OXDC and OXDC-CLEC are in the result that the stability in the presence of the trypsin is arranged as shown in Figure 9 after 0,2 and 5 hour.

The result of Fig. 9 shows that after 5 hours, OXDC-CLEC and uncrosslinked lenticular OXDC are stable at 37 ℃ of incubations, and can tolerate the Proteolytic enzyme cutting of chymase.The analysis showed that, compare that OXDC-CLEC and uncrosslinked lenticular OXDC enzyme keep about 100% activity with its initial value before 5 hours.Simultaneously, be exposed to chymase after 2 hours, about 50% activity of soluble proteins lose, after 5 hours, all soluble OXDC are degraded, and remain 0% activity.

Embodiment 22. soluble OXDC, lenticular OXDC and OXDC-CLEC are containing pancreatin Stability in the simulated intestinal fluid

Each 1mL simulated intestinal fluid that the OXDC-CLEC (embodiment 10) of the lenticular OXDC (embodiment 5) of soluble OXDC (embodiment 5), the 10.0mg/mL of 5.0mg/mL and 10.0mg/mL is placed in the Eppendorf tube (is recommended the preparation simulated intestinal fluid according to USB, the 6.8gm potassium dihydrogen phosphate is dissolved in 250ml water, mixes with 77ml 0.2N sodium hydroxide and 500ml deionization water; Then, add the 10gm pancreatin, pH is transferred to pH6.8 with 0.2N hydrochloric acid or 0.2N sodium hydroxide; Add water to 1L), at 37 ℃ of incubation 2h.Sampling in 0,1 and 2 hour, be used to measure the stability of enzyme.As described in embodiment 15, measure activity.The result as shown in figure 10.

Result shown in Figure 10 shows that OXDC-CLEC is stable in containing the simulated intestinal fluid of pancreatin, keep about 100% activity.On the contrary, most of soluble OXDC by pancreatin (mixture of lipase, amylase and protease) degraded, only remained about 26%-28% activity respectively behind incubation 1h and 2h in 1 hour.Having in the presence of the pancreatin, uncrosslinked lenticular OXDC is much more stable than its soluble form, only loses about 76% and about 49% activity behind incubation 1h and 2h respectively.

In addition, OXDC-CLEC meeting protective enzyme is avoided the cutting of protease (for example pepsin and chymase).Under the same conditions, the stability of OXDC-CLEC can be soluble OXDC stability at least about 100%, 200%, 300%, 400% or more.What under the same conditions, the soluble OXDC of specific activity that keeps of OXDC-CLEC kept is active high at least about 2,3 or 4 times.Thereby, compare with soluble OXDC, OXDC-CLEC the acid pH of scope from about 2.5 or 3 to about 7.5 or 8.5 pH and to contain under the intestinal harsh conditions of different protease be activated and be stable.

Many embodiments of the present invention have been described.However, should be appreciated that and to make different modifications, and do not break away from the spirit and scope of the present invention.Therefore, other embodiment also within the scope of the appended claims.

Claims (33)

1. comprise the crystalline pharmaceutical composition of oxalate decarboxylase.

2. the compositions of claim 1, wherein said crystal more has activity than the oxalate decarboxylase of soluble form.

3. the compositions of claim 2, wherein active high at pH 4 described crystalline specific activity soluble forms at least about 100%.

4. the compositions of claim 1, wherein said oxalate decarboxylase crystal is crosslinked with cross-linking agent, and have soluble form oxalate decarboxylase at least about 100% activity.

5. the compositions of claim 4, wherein said oxalate decarboxylase crystal is crosslinked with cross-linking agent, and have soluble form oxalate decarboxylase at least about 150% activity.

6. the compositions of claim 4, wherein said cross-linking agent is a glutaraldehyde.

7. the compositions of claim 6 wherein uses about 0.02% to the described oxalate decarboxylase crystal of about 1.5% (w/v) glutaraldehyde cross-linking.

8. the compositions of claim 7 is wherein used the described crystal of about 0.5% (w/v) glutaraldehyde cross-linking.

9. reduce the method for the oxalates in the mammal, this method comprise to described administration be enough to reduce described oxalates amount comprise the crystalline compositions of oxalate decarboxylase.

10. the method for claim 9 is wherein passed through the covalently bound described oxalate decarboxylase crystal of cross-linking agent.

11. the method for claim 10, wherein said crystal are activated in mammiferous gastrointestinal tract and are stable.

12. the method for claim 10, wherein said crystal is activated and is stable to about pH 8 at about pH 2.

13. the method for claim 10, wherein said compositions oral administration.

14. the method for claim 10, wherein said compositions is used for device outside.

15. the method for claim 10, the administration of wherein said compositions cause oxalates to be reduced by at least about 15%.

16. the method for claim 9, wherein said compositions oral administration.

17. measure the method for concentration of oxalate, this method comprises:

The preparation biological sample and

Use the concentration of oxalate in oxalate decarboxylase crystal or the crosslinked oxalate decarboxylase crystal measurement sample.

18. the method for the disease relevant with the concentration of oxalate that raises in the treatment mammal, this method comprises: be enough to reduce the oxalate decarboxylase crystal of the amount of one or more symptoms relevant with described disease for described administration.

19. the method for claim 18, wherein said disease is relevant with kidney or liver function.

20. the method for claim 18, wherein said disease is selected from: primary hyperoxaluria, intestinal originality hyperoxaluria, the special property sent out hyperoxaluria, ethylene glycol is poisoned, cystic fibrosis, inflammatory bowel, lithangiuria, renal calculus, chronic nephropathy, hemodialysis and gastrointestinal bypass.

21. make the crystalline method of albumen, this method comprises:

(a) in salinity dissolving or concentrate described albumen and

(b) reduce described salinity, form crystal.

22. the method for claim 21 is at least about 0.3M in salinity described in the step (a) wherein.

23. the method for claim 21 is about 0.5M in salinity described in the step (a) wherein.

24. the method for claim 21, wherein said salt is selected from sodium chloride and potassium chloride.

25. the method for claim 21, wherein said albumen is oxalate decarboxylase.

26. make albumen crystalline method from cell extract, this method comprises:

(a) to cell with salt, in salinity, form cell mixture and

(b) reduce described salinity, form crystal.

27. the method for claim 26 is at least about 0.3M in salinity described in the step (a) wherein.

28. the method for claim 26 is about 0.5M in salinity described in the step (a) wherein.

29. the method for claim 26, wherein said salt is selected from sodium chloride and potassium chloride.

30. the method for claim 26 is wherein with described cell mixture homogenization.

31. the method for claim 26, wherein said albumen is oxalate decarboxylase.

32. pass through the oxalate decarboxylase crystal of the method preparation of claim 21.

33. pass through the oxalate decarboxylase crystal of the method preparation of claim 22.

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