MXPA01001342A

MXPA01001342A - Urate oxidase

Info

Publication number: MXPA01001342A
Application number: MXPA/A/2001/001342A
Authority: MX
Inventors: Susan J Kelly; Michael Hershfield
Original assignee: Duke University; Michael Hershfield; Susan J Kelly
Priority date: 1998-08-06
Filing date: 2001-02-06
Publication date: 2002-02-26

Abstract

The present invention relates, in general, to urate oxidase (uricase) proteins and nucleic acid molecules encoding same. In particular, the invention relates to uricase proteins which are particularly useful as, for example, intermediates for making improved modified uricase proteins with reduced immunogenicity and increased bioavailability.

Description

URINE OXIDASE Field of the Invention The present invention relates, in general, to the urate oxidase (uricase) proteins and the nucleic acid molecules encoding them. In particular, the invention relates to uricase proteins that are particularly useful as, for example, intermediates for making improved modified uricase proteins with reduced immunogenicity and increased bioavailability. Preferred modified uricase proteinis of the present invention include the uricase proteins covalently linked with poly (ethylene glycols) or poly (ethylene oxides). The present invention therefore provides uricase proteins, antibodies that specifically bind to the proteins, nucleic acid molecules encoding the uricase proteins and useful fragments thereof, vectors containing the nucleic acid molecules, host cells containing the vectors and methods for using and making the uricase proteins and the nucleic acid molecules. Background Gout is the most common inflammatory disease of the joints in men older than 40 years (Roubenoff 1990). Painful gouty arthritis occurs when a high blood level of uric acid (hyperuricemia) leads to the episodic formation of microscopic crystals of monosodium urate monohydrate in the joints. Over time, chronic hyperuricaemia can also result in destructive crystalline urate deposits (tophi) around the joints, in soft tissues, and in some organs (Hershfield 1996). Uric acid has solubili < limited urine and when over-excreted (hyperuricosuria) can cause kidney stones (uricoli tiasis). In patients with certain malignancies, particularly leukemia and lymphoma, marked hyperuricemia and hyperuricosuria (due to increased tumor cell regress and lysis during chemotherapy) poses a serious risk of acute, obstructive renal failure (Sandberg et al., 1956; Fritz 1957; Cohen et al., 1980; Jones et al., 1990). Severe hyperuricaemia and gout are associated with renal dysfunction for several causes, including cyclosporine therapy to prevent allograft rejection of organs (West et al., 1987; Venkataseshan et al., 1990; Ahn et al., 1992; Delaney et al. 1992; George and Mandell 1995). Hyperuricemia can result from both overproduction of urate and subexcretion (Hershfield and Seegmiller 1976, Kelley and collaborators, 1989, Becker and Roessler 1995). When moderate, hyperuricaemia can be controlled with diet, but when pronounced and associated with serious clinical consequences, requires treatment with drugs, either a uricosuric substance that promotes the excretion of uric acid (ineffective if renal function is reduced) , or allopurinol inhibiting xanthine oxidase, which blocks the formation of urate. Allopurinol is the basis of therapy in patients with tophaceous gout, renal failure, leukemia, and some inherited disorders. The treatment for hyperuricemia is usually effective and well tolerated. However, some patients with disfiguring, disabling, tophaceous gout reject all conventional therapy (Becker 1988; Fam 1990; Rosenthal and Ryan 1995). Furthermore, approximately 2 percent of patients treated with allopurinol develop allergic reactions, and a severe hypersensitivity syndrome occurs in approximately 0.4 percent (Singer and Wallace 1986; Arellano and Sacristán 1993). This frequently life-threatening syndrome can cause acute renal and hepatic failure, and severe skin damage (epidermal necrolysis, exfoliative dermatitis, erythema multiforme Stevens-Johnson syndrome). Allopurinol also interferes with the metabolism of azathioprine and 6-mercaptopurine, drugs used in the treatment of leukemia and for the prevention of allograft rejection of organs, conditions in which marked hyperuricemia occurs and can cause severe gout or threaten function renal. Finally, hyperuricemia is the result of utational inactivation of the human gene for urate oxidase (uricase) during evolution (Wu et al., 1989; Wu et al., 1992). The active uricase in the peroxisomes of the liver of most non-human primates and other mammals converts urate to allantoin (+ C02 and H202), which is 80-100 times more soluble than uric acid and is "managed most efficiently by the kidney. Parenteral uricase, prepared from Aspergillus flavus (Uricozyme®, Clin-Midy, Paris), has been used to treat severe hyperuricaemia associated with leukemia chemotherapy for more than 20 years in France and Italy (London and Hudson 1957, Kissel et al., 1968; Brogard et al., 1972; Kissel et al., 1972; Potaux et al., 1975; Zittoun et al., 1976; Brogard et al., 1978; Masera et al., 1982), and has been used in recent clinical trials in leukemia patients in the United States of America. (Pui et al., 1997). Uricase has a faster onset of action than allopurinol (Masera et al., 1982, Pui et al., 1997). In patients with gout, uricase infusions can interrupt acute attacks and decrease the size of tophi (Kissel et al., 1968; Potaux et al., 1975; Brogard et al., 1978). Although effective in treating acute hyperuricemia during a short course of chemotherapy, the daily uricase infusion of A. flavus would be a serious disadvantage to treat recurrent gout or tophaceous. In addition, the efficacy of uricase A. flavus decreases rapidly in patients who develop anti-uricase antibodies (Kissel et al., 1968, Brogard et al., 1978; Escudier et al., 1984; Mourad et al., 1984; Sobiny et al., 1984). "Serious allergic reactions have occurred, including anaphylaxis (Donadío et al., 1981; Montagnac and Schillinger 1990; Pui et al., 1997). A longer, less immunogenic preparation preparation of uricase is clearly necessary for chronic therapy. One approach to sequester exogenous enzymes from proteases and the immune system involves the covalent attachment of the non-toxic, inert polymer, monomethoxy polyethylene glycol (PEG) to the surface of the proteins (Harris and Zalipsky 1997). The use of polyethylene glycols with Mr about 1,000 a > 10,000 was first shown to prolong circulating life and reduce the immunogenicity of several foreign proteins in animals (Abuchowski et al., 1977a, Abuchowski et al., 1977b, Davis et al., 1981a, Abuchowski et al., 1984, Davis et al., 1991). In 1990, bovine adenosine deaminase (ADA) modified with PEG of Mr 5000 (PEG-ADA, ADAGEN®, produced by Enzon, Inc.) became the first PEG-ized protein approved by the Food and Drug Administration of the United States. United, for the treatment of severe combined immunodeficiency disease due to ADA deficiency (Hershfield et al., 1987). Experience over the past 12 years has shown that anti-DAA antibodies can be detected by a sensitive ELISA test in most patients during chronic treatment with PEG-ADA, but there have been no allergic or hypersensitivity reactions; Accelerated elimination of PEG-ADA has occurred in some patients who produce anti-DAA antibodies, but this has usually been a transient effect (Chaffee et al., 1992; Hershfield 1997). It should be noted that the immune function of patients with ADA deficiency usually does not become normal by treatment with PEGADA (Hershfield 1995, Hershfield and Mitchell 1995). Thus, immunogenicity could be a more significant problem to develop a PEG-enzyme for the chronic treatment of patients with normal immune function. Immunogenicity will be understood by a person with ordinary experience as related to the induction of an immune response by an injected preparation of an antigen (such as modified protein PEG or unmodified protein), while antigenicity refers to the reaction of an antigen with previously existing antibodies. Collectively, antigenicity and immunogenicity are referred to as immunoreactivity. In previous studies of PEG-uricase, the immunoreactivity was assessed by a variety of methods, including: the in vitro reaction of PEG-uricase with previously formed antibodies; induced antibody synthesis measurements; and accelerated elimination rates after repeated injections. PEGization has been shown to reduce the immunogenicity and prolong the circulating life of fungal and porcine uricases in animals (Chen et al, 1981, Savoca et al., 1984; Tsuj i et al., 1985; Veronese et al., 1997). Candida uricase modified with PEG rapidly lowered serum urate to undetectable levels in five normouricemic human volunteers (Davis et al., 1981b). The PEG-ized Arthrobacter uricase produced by Enzon, Inc., was used in a compassionate base to treat a patient hypersensitive to allopurinol with lymphoma, who presented renal failure and marked hyperuricemia (Chua et al., 1988, Greenberg and Hershfield 1989). Four intramuscular injections were administered for approximately 2 weeks. During this brief period, hyperuricemia was controlled and no anti-uricase antibody could be detected by an ELISA test in the patient's plasma. The additional use and clinical development of this preparation was not followed. To date, no form of uricase or PEG-uricase having a conveniently long circulating life and sufficiently reduced immunogenicity for safe and reliable use in chronic therapy has been developed. The object of this invention is to provide an improved form of uricase which, in combination with the PEG-ization, can satisfy these requirements. The invention is an exclusive recombinant mammalian derivative uricase, which has been modified by mutation in a manner that has been shown to increase the ability of PEG-ization to mask the potentially immunogenic epitopes. SUMMARY OF THE INVENTION It is a general object of the present invention to provide novel uricase proteins and nucleic acid sequences encoding the same. It is another object of the present invention to provide a method for purifying recombinantly produced uricase proteins, such as those described herein. It is another object of the present invention to provide a method for reducing the amount of uric acid in a body fluid of a mammal by administering a composition containing a uricase protein of the present invention to the mammal. It is still another object of the present invention to provide antibodies to the uricase proteins described herein. It is another object of the present invention to provide vectors and host cells which contain nucleic acid sequences described herein and methods for using same to produce the uricase proteins encoded thereby. The present invention provides uricase proteins that can be used to produce a PEG-uricase not < substantially immunogenic that retains all or almost all the uricolytic activity of the unmodified enzyme. Uricolytic activity is expressed in the present in international units (Ul) per milligram of protein where an international unit of uricase activity is defined as the amount of enzyme that consumes one micromole of uric acid per minute. The present invention provides a recombinant uricase protein of a mammalian species that has been modified to insert one or more lysine residues. The recombinant protein, as used herein, refers to any artificially produced protein and is distinguished from naturally occurring proteins (i.e., that are produced in tissues of an animal that possesses only the natural gene for the specific protein of interest. ). The protein includes peptides and amino acid sequences. The recombinant uricase protein of the present invention can be a chimera or hybrid of 2 or 3 mammalian proteins, peptides or amino acid sequences. In one embodiment, the present invention can be used to prepare a recombinant uricase protein of a mammalian species, this protein has been modified to increase the number of lysine to the point that, after PEG-ization of the recombinant uricase protein, the PEG-ized uricase product is substantially as active enzymatically as the modified non-4 uricase and the PEG-ized uricase product is not unacceptably immunogenic. Truncated forms of uricases of the present invention are also contemplated in which the amino and / or carboxy termini of the uricase may not be present. Preferably, the uricase is not truncated to the extent that the lysines are removed. A person with ordinary experience will appreciate that the conjugate-carrier uricase complex may not contain as many linkages to substantially reduce the enzymatic activity of the uricase or too few bonds to remain unacceptably immunogenic. Preferably, the conjugate will retain at least about 70 percent to about 90 percent of the uricolytic activity of the unmodified uricase protein while being more stable, so that it retains its enzymatic activity during storage, in mammalian plasma. and / or serum at physiological temperature, in comparison with unmodified uricase protein. Retention of at least about 80 percent to about 85 percent of uricolytic activity would be acceptable. Moreover, in a preferred embodiment, the conjugate provides substantially reduced immunogenicity and / or immunoreactivity than the unmodified uricase protein. In one embodiment, the present invention provides a uricase protein described herein that can be modified by binding with a "non-toxic, non-immunogenic, pharmaceutically acceptable carrier, such as PEG, by covalent bonding with at least one of the lysines. contained in the uricase protein. Alternatively, the uricase protein is modified by covalently binding a carrier through less than about 10 lysins of its amino acid sequence. The binding to any of 2, 3, 4, 5, 6, 7, 8, or 9 of the lysines is contemplated as alternative modalities. The uricase protein of the present invention is a recombinant molecule that includes porcine liver and baboon uricase protein segments. A modified baboon sequence is also provided. In one embodiment, the present invention provides a chimeric pig-baboon uricase (PBC uricase (SEQ ID NO: 2)) that includes amino acids (aa) 1-225 of porcine uricase (SEQ ID NO: 7) and amino acids 226- 304 of baboon uricasa (SEQ ID NO: 6) (See also the sequence in Figure 5). In another embodiment, the present invention provides a pig-baboon uricase (uricase PKS) which includes amino acids 1-288 of porcine uricase and amino acids 289-304 of baboon uricase (SEQ ID NO: 4). The truncated derivatives of PBC and PKS are also contemplated. Preferred truncated forms are PBC and PKS proteins truncated either to suppress the amino terminal 6 amino acids or the 3 carboxy terminal amino acids, or both. The representative sequence is given in SEQ ID NO: 8 (PBC truncated to amino), 9 (PBC truncated to carboxy), 10 (PKS truncated to amino) and 11 (PKS truncated to carboxy). Each of the PBC uricase, PKS uricase and its truncated forms have from one to four more lysines than those found in other mammalian uricases that have been cloned. The present invention provides nucleic acid molecules (DNA and RNA sequences) that include nucleic acid molecules in isolated, purified and / or cloned form, which encode the uricase proteins and truncated proteins described herein. Preferred embodiments are shown in SEQ ID NO: 1 (uricase PBC) and SEQ ID NO: 3 (uricase PKS). Vectors (expression and cloning) that include these nucleic acid molecules are also provided by the present invention. Moreover, the present invention provides host cells containing these vectors. Antibodies that specifically bind to the uricase proteins of the present invention are also provided. Antibodies to the amino portion of the pig uricase and antibodies to the carboxy portion of the baboon uricase, when used together, should be useful for detecting PBC, or other similar chimeric proteins. Preferably, the antibody to the amino portion of the chimeric uricase should not recognize the amino portion of the uricase of the baboon and similarly, the antibody to the carboxy portion of the chimeric uricase should not recognize the carboxy portion of the pig uricase. More preferably, antibodies are provided that specifically bind PBC or PKS but do not bind to natural proteins, such as pig and / or baboon uricases. In another embodiment, the present invention can be used to prepare a pharmaceutical composition for reducing the amount of uric acid and body fluids, such as urine and / or serum or plasma, containing at least one of the uricase proteins. or uricase conjugates described herein and a pharmaceutically acceptable carrier, diluent or excipient. The present invention can also be used in a method for reducing the amount of uric acid in the body fluids of a mammal. The method includes administering to a mammal an amount effective to lower the uric acid of a composition containing a uricase protein or a uricase conjugate of the present invention and a diluent, carrier or excipient, which preferably is a carrier, diluent or excipient. pharmaceutically acceptable excipient. The mammal to be treated preferably is a human. The administration step can be, for example, injection by intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal routes. Elevated uric acid levels may be in blood or urine, and may be associated with gout, tophi, kidney failure, organ transplantation, or malignancy. In another embodiment, the present invention provides a method for isolating and / or purifying a uricase from a uricase solution containing, for example, cellular and subcellular debris from, for example, a recombinant production process. Preferably, the purification method takes advantage of the limited solubility of mammalian uricase with low pH (Conley et al., 1979), washing the crude recombinant extract with a pH of about 7 to about 8.5 to remove a majority of the proteins that are soluble. in this low pH range, after which the active uricase is solubilized in a regulator, preferably sodium carbonate buffer, at a pH of about 10-11, preferably about 10.2. The solubilized active uricase can then be applied to an anion exchange column, such as a Q Sepharose column, which is washed with a low or high salt gradient in a regulator at a pH of about 8.5, after which the purified uricase eluting with a gradient of sodium chloride in a sodium carbonate buffer at a pH of about 10 to about 11, preferably about 10.2. The enzyme can be further purified by gel filtration chromatography at a pH of about 10 to about 11. In this step, the enzyme can be further purified by lowering the pH to about 8.5 or less to selectively precipitate uricase, but no more contaminants soluble. After washing at low pH (7-8) the uricase is solubilized at a pH of about 10.2. The preparation of uricase could be analyzed by methods known in the art of pharmaceutical preparation, such as, for example, any of the high performance liquid chromatography (HPLC), other chromatographic methods, light scattering, centrifugation and / or electrophoresis. in gel. Brief description of the drawings Figure 1. Analysis SDS-mercaptoethanol PAGE (12% gel) Figure 2. Circulating life of natural PBC uricase and PEG-ized. Figure 3. Relationship of uricase activity in serum with serum and urine concentrations of uric acid. Figure 4. Maintenance of the circulating level of uricase activity (measured in serum) after repeated injection.

Figure 5 shows the amino acid sequences deduced from the chimeric pig-baboon uricase (uricasa PBC) and porcine uricase containing the R291K mutations and T301S (uricase PKS), compared with porcine and baboon sequences. Figure 6. Comparison of the amino acid sequence PKS and pig uricase. Figure 7. Comparison of the amino acid sequence of PBC and PKS. Figure 8. Comparison of the amino acid sequence of PBC and pig uricase. Figure .9. Comparison of the amino acid sequence of pig uricase and D3H. Figure 10. Comparison of amino acid sequence of PBC and D3H. Figures 11-1 and 11-2. Fit comparison (GCG software) of PKS uricase and pig cDNA coding sequences. Figures 12-1 and 12-2. Comparison of fit (GCG software) of the coding sequences of the PKS cDNAs and baboon uricase. Figures 13-1 and 13-2. Comparison of fit (GCG software) of coding sequences of PBC cDNAs and pig uricase. Figures 14-1 and 14-2. Comparison of fit (GCG software) of the coding sequences of PBC cDNAs and baboon uricase. DETAILED DESCRIPTION OF THE INVENTION The present invention provides uricase proteins which are useful intermediates for improved uricase conjugates of water soluble polymers, preferably poly (ethylene glycols) or poly (ethylene oxides), with uricases. Uricase, as used herein, includes individual subunits as well as the natural tetramer, unless otherwise indicated. Although humans do not make an active enzyme, transcripts of uricase mRNA have been amplified for human liver RNA (Wu et al., 1992). Theoretically it is possible that some transcripts of human uricase are translated; even if the peptide products did not have full length or were unstable, they could be processed by antigen-presenting cells and play a role in determining the immune response to an exogenous uricase used for the treatment. In theory, it may be possible to reconstruct and express a human uricase cDNA by eliminating the two known nonsense mutations. However, in the absence of selective pressure, it is very likely that deleterious nonsense mutations have accumulated in the human gene during the millions of years since the first nonsense mutation was introduced (Wu et al., 1989; Wu et al., 1992). ). Identifying and "correcting" all mutations to obtain maximum catalytic activity and maximum protein stability would be very difficult. The present inventors have appreciated that there is a high degree of homology (similarity) between the amino acid sequence deduced from human uricase with that of pig (approximately 86 percent) and baboon (approximately 92 percent) (see, Figure 6-14, as an example of similarity measurement), while the homology (similarity) between human uricase and that of A. flavus is < 40 percent (Lee et al., 1988; Reddy et al., 1988; Wu et al., 1989; Legoux et al., 1992; Wu et al., 1992). The present invention provides chimeric uricase proteins produced recombinantly from two different mammals that have been designed to be less immunoreactive to humans than the related fungal or bacterial enzyme more distantly. The use of mammalian uricase derivative is expected to be more acceptable to patients and their physicians. Experience has shown that activated PEGs such as those that have been used to make PEG-ADA and to modify other bound proteins via primary amino groups of the amino terminal residue (when present and not blocked) and epsilon-amino groups of lysines. This strategy is useful both because weak reaction conditions can be used, and because the positively charged glycines tend to be located on the surface of the proteins. The latter is important since for any therapeutic protein the desired effects of PEG-ization will depend in part on the characteristics of the PEG polymer (eg, mass, "branched or unbranched structure, etc.) as well as the number and distribution of the PEG binding sites of the protein in relation to the epitopes and the structural elements that determine the function and elimination of the protein. A strategy to increase the capacity of PEG-ization for "Masking" epitopes and reducing immunogenicity by semi-selective introduction of novel lysine residues for the potential addition of polyethylene glycol has been considered (Hershfield et al., 1991). This strategy employs mutagenesis to replace the selected arginine codons with lysine codons, a substitution that maintains the positive charge and has minimal effect on the computer predicted indices of surface probability and antigenicity (useful when only the amino acid sequence is known). . As an experimental test of this strategy, purine nucleoside phosphorylase from recombinant E. coli has been used (EPNP) (Hershfield et al., 1991). The substitutions Arg-por-Lys at 3 sites were introduced, increasing the number of lysines per subunit from 14 to 17, without altering the catalytic activity. The purified triple mutant retained complete activity after the modification of approximately 70 percent of the accessible NH2 groups with excess of disuccinyl-PEG5000. Titration of the reactive amino groups before and after PEG-ization suggested that the triple mutant could accept one more chain of PEG per subunit than the enzyme of the original type. PEG-ization increased the circulating life of both the original and mutant type of EPNP enzymes in mice from about 4 hours up to > 6 days . After a series of intraperitoneal injections at weekly / biweekly intervals, all mice treated with both unmodified EPNP, and 10 of 16 mice (60 percent) injected with original PEG-ized EPNP type, developed high levels of anti-EPNP antibody with a marked decline in the circulating life. In contrast, only 2/12 mice (17 percent) treated with PEG-EPNP developed rapid elimination; Low antibody levels in these mice do not correlate with circulating life. This strategy was successful in substantially reducing immunogenicity although only 1 of 3 new lysins became modified after treatment with activated PEG. The uricasa subunits of baboon and pig each consist of 304 amino acids, 29 of which (ie, 1 in approximately 10 residues) are lysines. Initial attempts to introduce substitutions of Arg-por-Lys in the cDNA for the baboon uricase, and also a substitution of Lys for a Glu codon at position 208, which is known as a Lys in the human uricase gene, gave as a result an expressed mutant baboon protein that greatly reduced the catalytic activity of uricase. It was apparent from this experiment the ability to maintain the activity of the enzyme uricase after the mutation of arginine into lysine from the mammalian DNA sequence was not predictable. Subsequently, it was found that the amino acid residue 291 in the uricase of baboon is lysine, but the corresponding residue in pig is arginine. The Apal restriction site present in both cDNAs was exploited to construct a chimeric uricase in which the first 225 amino acids are derived from the pig cDNA and the carboxy terminal 79 are derived from the baboon cDNA. The uricase (PBC) chimeric pig-baboon (SEQ ID N0: 2) has 30 lysines, one more than any "parent" enzyme. An additional feature of the PBC uricase is that the "baboon" portion differs from the human uricase in 4 of 79 amino acid residues, while the pig and human uricase differ by 10 in the same region. A modified version of PBC was subsequently constructed, which maintains the extra lysine at position 291 and otherwise differs from pig uricase only by a substitution of serine by threonine at residue 301 (uricasa "pig KS" (SEQ ID NO. :4)). In view of the results described in the previous paragraph within several other lysine insertions were detrimental to the activity, it was unexpected that the chimeric uricase PBC and PKS it was completely as active as compared to the original undiutated pig uricasa and approximately more than four times as active as the original, unmutated baboon uricasa. The present invention provides a chimeric pig-baboon uricase composed of portions of the uricase sequences of pig liver and baboon. An example of this chimeric uricase contains the first 225 amino acids of the porcine uricase sequence (SEQ ID NO: 7) and the last 79 amino acids of the baboon uricase sequence (SEQ ID NO: 6) (pig-baboon uricasa), or uricasa PBC; Figure 6 and SEQ ID NO: 2). Another example of this chimeric uricase contains the first 288 amino acids of the porcine sequence (SEQ ID NO: 7) and the last 16 amino acids of the baboon sequence (SEQ ID NO: 6). Since the last sequence differs from the porcine sequence in only two positions, having a lysine (K) in place of arginine at residue 291 and a serine (S) instead of a threonine at residue 301, this mutant is known as uricasa of pig-KS or PKS. Vectors (expression and cloning) that include nucleic acid molecules encoding the proteins of the present invention are also provided. Preferred vectors include those exemplified herein.

A person with ordinary experience will appreciate that nucleic acid molecules can be inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the nucleic acid (DNA) can be ligated with suitable transcription and translation nucleotide sequences recognized by the desired host, although these control elements are generally available in the expression vectors used and known in the art. The vector can then be introduced into the host cells by standard techniques. Generally, not all host cells are transformed by the vector. It may be necessary, therefore, to select the transformed host cells. A selection method known in the art involves incorporating into the expression vector a DNA sequence, with any necessary control element, that encodes a selectable marker trait in the transformed cell, such as resistance to the antibiotic. Alternatively, the gene for this selectable trait may be in another vector which is used to cotransform the desired host cells. The vectors may also include a suitable promoter, such as a prokaryotic promoter capable of expression (transcription and translation) of the DNA in a bacterial host cell, such as E. coli, transformed therewith. Many expression systems are available and are known in the art, including bacterial cells (e.g., E. coli and Bacillus subtili s), yeasts (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus), plant cells, animal cells and insect cells. Suitable vectors may include a prokaryotic replica, such as ColEl ori, for propagation in, for example, a prokaryote. Prokaryotic vector plasmids are pUC18, pUC19, pUC322 and pBR329 available from Biorad Laboratories (Richmond, CA) and pTcr99A and pKK223-3 available from Pharmacia (Piscataway, NJ). A typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, NJ). This vector uses the last SV40 promoter to drive the expression of cloned genes, the highest level of expression is found in cells that produce T antigen, such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the inducible glucocorticoid promoter of the long terminal repeat of the mouse mammary tumor virus to drive the expression of the cloned gene. Useful yeast plasmid vectors are pRS403-406 and pRS413-416, and are generally available from Stratagene Cloning Systems (LaJolla, CA). The plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integration plasmids (Yips) and incorporate the selectable yeast markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are yeast centomer plasmids (Ycps). Moreover, the present invention provides host cells containing these vectors. Preferred host cells include those exemplified and described herein. The uricase proteins of the present invention can be conjugated via a non-toxic, biologically stable, covalent bond with a relatively small number of PEG chains to improve the biological half-life and solubility of the proteins and reduce their immunoreactivity. These bonds may include urethane (carbamate) linkages, secondary amino linkages, and amide linkages. Various activated polyethylene glycols suitable for this conjugation are commercially available from Shearwater Polymers, Huntsville, AL. The invention can also be used to prepare pharmaceutical compositions of uricase proteins as conjugates. These conjugates are substantially non-immunogenic and retain at least 70 percent, preferably 80 percent, and more preferably at least about 90 percent or more of the uricolytic activity of the unmodified enzyme. Water-soluble polymers suitable for use in the present invention include poly (ethylene glycols) or linear and branched poly (ethylene oxides), all commonly known as PEGs.

An example of branched PEG is the subject of U.S. Patent No. 5,643,575. In one embodiment of the invention, the average number of lysins inserted per uricase subunit is between 1 and 10. In a preferred embodiment, the number of additional lysines per uricase subunit is between 2 and 8. It is understood that the number of lysines additional must not be so large to be detrimental to the catalytic activity of the uricase. The PEG molecules of the conjugate are preferably conjugated through the lysins of the uricase protein, more preferably, through a non-naturally occurring lysine or lysines, which have been introduced into the portion of a designated protein that It does not naturally contain a lysine in that position. The present invention provides a method for increasing the non-detrimental PEG binding sites available to an uricase protein wherein the original uricase protein is mutated so as to introduce at least one lysine residue therein. Preferably, this method includes the replacement of arginines with lysines. PEG-uricase conjugates using the present invention are useful for lowering the levels (i.e., reducing the amount) of uric acid in the blood and / or urine of mammals, preferably humans, and thus can be used for the treatment of elevated levels of uric acid associated with conditions that include gout,. tophi, renal failure, organ transplantation and malignant disease. The PEG-uricase conjugates can be introduced into a mammal having excessive levels of uric acid by any of several routes, including oral, by enema or suppository, intravenous, subcutaneous, intradermal, intramuscular and intraperitoneal routes. Patton, JS, et al., (1992) Adv Drug Delivery Rev 8: 179-228. The effective dose of PEG-uricase will depend on the level of uric acid and the size of the individual. In one embodiment of this aspect of the invention, the PEG-uricase is administered in a pharmaceutically acceptable excipient and diluent in an amount ranging from 10 micrograms to about 1 gram. In a preferred embodiment, the amount administered is between about 100 micrograms and 500 milligrams. More preferably, the conjugated uricase is administered in an amount between 1 milligram and 100 milligrams, such as, for example, 5 milligrams, 20 milligrams, or 50 milligrams. The masses given for the dose amount of the modalities refer to the amount of protein in the conjugate. Pharmaceutical formulations containing PEG-uricase can be prepared by conventional techniques, for example, as described in Remington Pharmaceutical Sciences, (1985) Easton, PA: Mack Publishing Co. Suitable excipients for the preparation of injectable solutions include, example, phosphate buffered saline solution, lactated Ringer's solution, water, polyols and glycerol. Pharmaceutical compositions for parenteral injection comprise liquids, dispersions, suspensions, emulsions "aqueous or non-aqueous as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just before use. These formulations may contain additional components, such as, for example, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, regulators, antioxidants and diluents. PEG-uricase can also be provided as implant-controlled release compositions in an individual to continuously monitor high levels of uric acid in blood and urine. For example, polylactic acid, polyglycolic acid, regenerated collagen, poly-L-lysine, sodium alginate, gellan gum, chitosan, agarose, multilamellar liposomes and many other conventional deposit formulations comprise bioerodible or biodegradable materials that can be formulated with biologically active compositions. These materials when implanted or injected, they gradually disintegrate and release the active material to the surrounding tissue. For example, a method for encapsulating PEG-uricase comprises the method described in U.S. Patent No. 5,653,974, which is incorporated herein by reference. The use of bioerodible, biodegradable formulations and other deposit formulations is expressly contemplated in the present invention. The use of infusion pump and matrix trapping system for the administration of PEG-uricase is also within the scope of the present invention. PEG-uricase can also advantageously be enclosed in micelles or liposomes. The liposome encapsulation technology is well known in the art. See, for example, Lasic, D, et al., (Eds.) (1995) Stealth Liposomes, Boca Raton, FL: CRC Press. The PEG-uricase pharmaceutical compositions described herein will decrease the need for hemodialysis in patients at high risk of urate-induced renal failure, eg, organ transplant recipients (see Venkataseshan, VS, et al., (1990) Nephron 56 : 317-321) and patients with some malignancies. In patients with large accumulations of crystalline urate (tophi), these pharmaceutical compositions will improve the quality of life more rapidly than currently available treatments. The following examples, which are not considered to be limiting of the invention in any way, illustrate the different aspects described above.

EXAMPLE 1 A. Construction of related PBC, PKS and uricase cDNAs Standard methods were used, and where applicable with instructions supplied by the reagent manufacturers, to prepare total cellular RNA, for amplification in polymerase chain reaction (Patents of United States of America numbers 4,683,195 and 4,683,202, 4,965,188 and 5,075,216) of urate oxidase cDNA, and for cloning and sequencing these cDNAs (Erlich 1989; Sambrook et al., 1989; Ausubel 1998). Polymerase chain reaction primers for pig and baboon urate-oxidases (Table 1) were designed based on published coding sequences (Wu et al., 1989) and using the PRIME software program (Genetics Computer Group, Inc.) . Table 1. Primers for the PCR amplification of urate oxidase cDNA of pig liver uricase cDNA: sense: 5 'gcgcgaattccATGGCTCATTACCGTAATGACTACA 3'. | Antisense: 5 'gcgctctagaagcttccatggTCACAGCCTTGAAGTCAGC 3'. | AUNÓ de'Uridása of baboon liver U3TT sense: 5 'gcgcgaattccATGGCCCACTACCATAACAACTAT 3' jantisentido: 5 'gcgcccatggtctagaTCACAGTCTTGAAGACAACTTCCT I The restriction enzyme sequences (lower case) introduced at the ends of the primers and sense (pig and baboon) EcoRI and Ncol; antisense (pig) Ncol, HindIII, Xbal; antisense (baboon) Ncol. In the case of the baboon sense primer, the third GAC codon (Aspartate) present in the baboon urate oxidase (Wu et al., 1992) was replaced with CAC (Histidine), the codon that is present in this position in the sequence of coding of the human urate oxidase pseudogene (Wu et al., 1992). For this reason I read recombinant baboon urate oxidase generated from the use of these primers has been named baboon urate-oxidase D3H. The total cellular RNA of the pig and baboon livers was reverse transcribed using a first chain kit (Pharmacia Biotech Inc. Piscataway, NJ). Amplification by polymerase chain reaction using Taq DNA polymereisate (GibcoBRL, Life Technologies, Gaithersburg, MD) was performed in a thermal cycler (Ericomp, San Diego, CA) with the program [30 s, 95 ° C; 30 s, 55 °; 60 s, 70o], 20 cycles, followed by [30 s, 95 ° C; 60 s, 70o] 10 cycles. The urate oxidase polymerase chain reaction products were digested with EcoRI and HindIII and cloned into pUC18 (pig), and also directly cloned (pig and baboon D3H) using the TA cloning system (Invitrogen, Carlsbadm CA). CDNA clones were transformed into the E. coli XLl-Blue chain (Stratagene, La Jolla, CA). Plasmid DNA containing cloned uricase cDNA was prepared and the sequence of the cDNA insert was analyzed by standard dideoxy technique. Clones possessing the published urate oxidase DNA coding sequence (except for the D3H substitution in baboon urate oxidase described in Table I) were constructed and verified in a series of subsequent steps by standard recombinant DNA methodology . The pig and baboon D3H cDNAs containing the full-length coding sequence were introduced into pET expression vectors (Novagen, Madison, Wl) as follows. The bacillus uricase D3H cDNA was separated from the TA plasmid with the restriction enzymes Ncol and BamHI and then subcloned into the Ncol and BamHI cloning sites of the expression plasmids pET3d and pET9d. The full-length pig uricase cDNA was separated from a pUC plasmid clone with the restriction enzymes EcoRI and HindIII and subcloned into the EcoRI and HindIII sites of pET28b. The coding region of the pig cDNA was also introduced into the Ncol and Blpl sites of the expression plasmid pET9d after separation of the Ncol and Blpl sites from pET28b. The pig-baboon chimera (PBC) cDNA was constructed by separating the 624 base pair Ncol-Apal restriction fragment from the bacilli uricase D3H cDNA from a baboon clone pET3d-D3H, and then this segment of baboon D3H was replaced with the restriction fragment 624 base pairs Ncol-Apal of pig cDNA. The resulting PBC urate oxidase cDNA consists of the pig urate oxidase codons 1-225 bound in frame with codons 226-304 of the baboon urate oxidase. The pig urate-oxidase-KS cDNA (pigKS) was constructed by separating the 864 base pair Ncol-Ndel restriction fragment from the baboon uricase D3H cDNA from a baboon pET3d-D3H clone, and then replacing this baboon segment D3H with the restriction fragment of 864 base pairs Ncol-Ndel from the pig cDNA. The resulting PKS urate oxidase cDNA consists of the codons of pig urate oxidase 1-288 bound in frame with codons 289-304 of baboon urate oxidase. The amino acid sequence of baboon urate oxidase D3H, PBC, and PKS are shown in Figure 5 and SEQUENCE LISTING. Standard techniques were used to prepare 15 percent glycerol materials for each of these transformants, and these were stored at -70 ° C. Where each of these species was expressed and the recombinant enzymes were isolated (Table 2), the pig, the PBC chimera, and the pork uricasas had very similar specific activity, which was approximately 4-5 times higher than the specific activity of recombinant baboon uricase. This order was confirmed in varicose other experiments. The specific activity of the PBC uricase prepared by several different procedures varied over a range of 2-2.5 times.

Table 2: Comparison of uricase from expressed recombinant mammals * The protein was determined by the Lowry method. Uricase activity was determined spectrophotometrically (Priest and Pitts 1972). The test was carried out at 23-25 ° C in a 1 cm quartz cuvette containing 1 milliliter reaction mixture (0.1 M sodium borate, pH 8.6, 0.1 mM uric acid). The disappearance of uric acid was monitored by the decrease in absorbance at 292 nm. An international unit (Ul) of uricase catalyzes the disappearance of one μmol of uric acid per minute. The E.coli transformants BL21 (DE3) pLysS of the four cDNA-pET uricases constructs indicated in Table 2 were plated on LB agar containing several antibiotics (carbenicillin and chloramphenicol for pET3d (pigKS); kanamycin and chloramphenicol for pET9d (PBC, pig, baboon)), as directed in the pET System Manual (Novagen, Madison Wl). 5 milliliter cultures (LB plus antibiotics) were inoculated with single transformant colonies and cultured for 3 hours at 37 ° C. Aliquots of 0.1 milliliter were then transferred to 100 milliliters of LB medium containing selective antibiotics and 0.1 percent of lactose (to induce uricase expression). After overnight development at 37 ° C, the bacterial cells from the aliquots of 0.5 milliliters of the cultures were extracted in charge controller SDS-PAGE, and analyzed by SDS-mercaptoethanol PAGE; this established that comparable levels of uricase protein had been expressed in each of the four cultures (the results are not shown). The remaining cells of each 100 milliliter culture were harvested by centrifugation and washed in PBS. The cells were resuspended in 25 milliliters of phosphate buffered saline, pH 7.4 (PBS) containing 1 mM of protease inhibitor AEBSF (Calbiochen, San Diego, CA) and then lysed on ice in a Bacterial Ceel Disruptor (Microfluidics, Boston MA). The insoluble material (including uricase) was agglomerated by centrifugation (20,190 x g, 4o, 15 min). The agglomerates were washed twice with 10 milliliters of PBS, and then extracted overnight at 4 ° C with 2 milliliters of 1 M Na 2 CO 3, pH 10.2. The extracts were diluted to 10 milliliters with water and then centrifuged (20.190 x g, 4o, 15 min). The uricase activity and protein concentrations were then determined.

EXAMPLE 2 Expression and isolation of recombinant PBC uricase (preparation in fermentor 4 liters) The uricase transformant pET3d-PBC was plated from a stock of glycerol. Erol on an LB agar plate containing carbenicillin and chloramphenicol, according to the directions of the Novagen pET system manual. A 200 milliliter inoculum started from a single colony was prepared in liquid medium of LB antibiotic on a rotary shaker (250 RPM) at 37 °, using procedures recommended in the pET system manual to maximize the retention of plasmid pET. At an OD525 of 2.4, cells from this 200 milliliter culture was collected by centrifugation and resuspended in 50 milliliters of fresh medium. This suspension was transferred to a high density fermenter containing 4 liters of SLBH medium containing carbenicillin and chloramphenicol (The composition of the SLBH medium, and the design and operation of the fermentor are described in (er et al., 1974)).

After 20 hours of cultivation under oxygen at 32 ° C (OD525 = 19), isopropylthiogalactoside (IPTG) was added at 0.4 mM to induce uricase production. After 6 more hours (OD525 = 37) bacterial cells were harvested by centrifugation (10.410 x g, 10 min, 4 ° C), washed once with PBS, and stored frozen at -20 ° C. Bacterial cells (189 grams) were resuspended in 200 milliliters PBS and lysed at the same time as cooling in an ice / salt bath by sonication (Heat Systems Sonicator XL, CL probe model, Farmingdale, NY) for 4 x 40 seconds in bursts at 100 percent intensity, with one minute of rest between bursts. The insoluble material in PBS (including the uricase) was pelleted by centrifugation (10,410 x g, 10 min, 4 ° C), and then washed 5 times with 200 milliliters of PBS. The uricase in the PBS insoluble agglomerate was extracted in 80 milliliters of 1 M Na 2 CO 3, pH 10.2 containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 130 micrograms / ml of aprotinin. The insoluble waste was removed by centrifugation (20,190 x g, 2 hours, 4 ° C). All additional steps in the purification were done at 4 ° C (the results are summarized in Table 3). The extract with Ph 10.2 was diluted in 1800 milliliters with 1 mM PMSF (to reduce Na2C03 to 0.075 M). This was applied to a column (2.6 x 9 cm) of fresh Q-Sepharose (Pharmacia Biotech, Inc., Piscataway, NJ), which has been equilibrated with 0.075 M Na2CO3, pH 10.2. After loading, the column was washed successively with 1) 0.075 M Na 2 CO 3, pH 10.2 until the absorbance A280 of the effluent reached the bottom; 2) 10 mM NaHCO3, pH 8.5 until the pH of the effluent fell to 8.5; 3) 50 ml of 10 mM NaHCO 3, pH 8.5, 0.15 M NaCl; 4) a gradient of 100 ml of 0.15 M up to 1.5 M NaCl in 10 mM NaHCO 3, pH 8.5; 5) 150 ml of 10 mM NaHCO 3, pH 8.5, 1.5 M NaCl; 6) 10 mM NaHCO 3, pH 8.5; 7) 0.1 MN? 2C03, pH 11 until the pH of the effluent was raised to 11. Finally, the uricase was eluted with a gradient of 500 milliliters of 0 to 0.6 M NaCl in 0.1 M Na 2 C0 3, pH 11. The activity eluted in two absorption peaks A280, which were placed separately (Fraction A and Fraction B, Table 3). The uricase in each of these deposits was precipitated by lowering the pH to 7.1 by the slow addition of 1 M acetic acid, followed by centrifugation (7,000 x g, 10 min). The resulting agglomerate was dissolved in 50 milliliters of 1 M Na 2 CO 3, pH 10.2 and stored at 4 ° C. Table 3 Purification of recombinant baboon-chimeric uricase (PBC) uricase IPTG-induced cell paste = 189.6 g * The uricase present in fraction A began to precipitate spontaneously after elution of the column. Therefore the activity measured in this purification step was underestimated. EXAMPLE 3 Small scale preparation and PEGylation of recombinant PBC uricase. This example shows that the purified recombinant PBC uricase can be used to produce PEG-ilada uricase. In this reaction, all the uricase subunits were modified (Figure 1, lane 7), with retention of approximately 60 percent catalytic activity (Table 4). A. Small scale expression and isolation of PBC uricase (Table 4, Figure 1). A 4 liter culture of E. coli BL21 (DE3) pLysS transformed with pET3d-PBC cDNA was incubated on a rotary shaker (250 rpm) at 37 °. At 0.7 OD525, the culture was induced with IPTG (0.4 mM, 6 hours). The cells were harvested and frozen at -20 ° C. The cells (15.3 grams) were disrupted by freezing and thawing, and extracted with 1 M Na2CO3, pH 10.2, 1 mM PMSF. After centrifugation (12,000 xg, 10 min, 4 ° C) the supernatant (85 milliliters) was diluted 1:10 with water and then chromatographed on Q-Sepharose in a manner similar to that described in Example 1. The activity uricase deposited from this step was concentrated by pressure ultrafiltration using a PM30 membrane (Amicon, Beverly, MA). The concentrate was chromatographed on a column (2.5 x 100 cm) of Sephacryl S-200 (Pharmacia Biotech, Piscataway, NJ) which was equilibrated and run in 0.1 M Na2CO3, pH 10.2. Fractions containing uricase activity were deposited and concentrated by pressure ultrafiltration, as above. B. PEG-ilación. 100 milligrams of concentrated Sephacryl S-200 PBC uricase (5 milligrams / milliliter, 2.9 μmol enzyme, 84.1 μmol lysine) in 0.1 M Na 2 CO 3, pH 10.2 was allowed to react with a 2-fold excess (mole of lyses uricase PEG: mol) of an activated form of PEG at 4o for 60 minutes. The PEG-ilada uricase was released from any unreacted or hydrolyzed PEG by tangential flow diafiltration. In this step the reaction was diluted 1:10 in 0.1 M Na2CO3, pH 10.2 and diafiltered versus 3.5 vol 0.1 M Na2CO3, pH 10.2, then against 3.5 vol 0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2. The filter sterilized enzyme was stable at 4o for at least one month. Table 4 Summary of purification and PEG-ilation of recombinant pig-baboon chyma uricase (PBC) Figure 1 shows an SDS-mercaptoethanol PAGE analysis (12 percent gel) of fractions obtained during purification and PEGylation of recombinant baboon-pig chimera (PBC) uricase. Strips: 1 = MW markers; 2 = SDS SDS extract of uninduced pET3d-PBC cDNA transformed cells (E. coli BL21 (DE3) pLysS); 3 = SDS extract of cells transformed by pET-PBC cDNA induced by IPTG; 4 = crude extract (see Table 5); 5 = reservoir of concentrated Q-sepharose uricase; 6 = deposit of uricase Sephacril S-200 concentrated; 7 = recombinant PBC uricase Sephacryl S-200 PEG-ilada. "The results shown in Table 4 show that purified PBC uricase could be modified with retention of approximately 60 percent catalytic activity. In this PEGylation reaction all uricase subunits were modified (Figure 1, lane 7). In studies not shown, the PEG-ized enzyme had kinetic properties similar to the unmodified PBC uricase (K "10-20 μM). Importantly, the modified enzyme is much more soluble than the unmodified enzyme at physiological pH (> 5 mg / ml in PBS against < 1 mg / ml). The PEG-ized enzyme could also be lyophilized and then also reconstituted in PBS, pH 7.2, with minimal loss of activity. In other experiments, we compared the preparation activities of uricase PBC-PEG with the uricase clinical preparation of A. flavus At pH 8.6 in borate regulator, the enzyme of A. flavus had 10-14 times higher Vmax and a KM 2 times higher. However, in PBS, pH 7.2, PEG-PBC and unmodified fungal enzymes differed in uricase activity by <2 times EXAMPLE 4 Circulating life of PBC uricase unmodified and PEG-ized in mice. Figure 2 shows the circulating life of original PBC uricase and PEG-ized. Groups of mice (3 per point in time) were injected intraperitoneally with an original recombinant PBC uricase unit (circle) or modified with PEG (frames) (preparation described in Example 3). At the indicated times, blood was obtained from sets of three mice to measure uricase activity in serum. The PEG-ized uricase (described in Example 3) had a circulating half-life of approximately 48 hours, against < 2 hours for the unmodified enzyme (Figure 2). EXAMPLE 5 Efficacy of the PEG-ized uricase of the invention Figure 3 shows the ratio of uricase activity in serum to uric serum and urine concentrations. In this experiment, a homozygous mouse with genes cut out deficient in uricase (Wu et al., 1994) received two injections, at 0 and 72 hours, of 0.4 UI of uricase Recombinant PBC that was PEG-ized. The uricase deficient knockout mouse was used in this experiment because, unlike normal mice that had uricase, these knockout mice, like humans, had high levels of uric acid in their blood and body fluids and They excrete high levels of uric acid in their urine. These high levels of uric acid caused serious damage to the kidneys of these mice, which is frequently fatal (Wu et al., 1994). The experiment shown in Figure 3 demonstrates that intraperitoneal injections of a PEG-ized preparation of uricosease PBC recombinant resulted in an increase in uricase activity in serum, which was accompanied by a marked decline in serum and urinary concentrations of uric acid in a mouse with uricase deficiency. EXAMPLE 6 Lack of immunogenicity of construction-carrier complex PEG-ized recombinant PBC uricase was repeatedly injected into mice with uricase deficiency without inducing accelerated elimination, consistent with absence of significant immunogenicity. This was confirmed by the ELISA test. Figure 4 shows the maintenance of circulating levels of uricase activity (measured in serum) after repeated injection. The PEG-ized uricase PBC was administered by intraperitoneal injection at 6-10 day intervals. 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Role of anti-serum urate oxidase precipitating antibodies (letter). Presse Med 13: 443 Singer JZ, Wallace SL (1986) The allopurinol hypersensitivity syndrome. Unnecessary morbidity and mortality. Arthritis Rheum 29: 82-87 Tsuj i J, Hirose K, Kasahara E, Naitoh M, Yamamoto I (1985) Studies on the antigenicity of the polyethylene glycol-modified uricase. Int J I munopharmacol 7: 725-730 Vankataseshan VS, Feingold R, Dikman S, Churg J (1990) Acute hyperuricemic nephropathy and renal failure after transplantation. Nephron 56: 317-321 Veronese FM, Caliceti P, Schiavon O (1997) New synthetic polymers for enzyme and liposome modification. In: Haris JM, Zalipsky S (eds) Poly (ethylene glycol) Chemistry and Biological Applications, ACS, Washington, DC, ppl82-192 West C, Carpenter BJ, Hakala TR (1987) The incidence of gout in renal transplant recipients. Am J Kidney Dis 10: 369-371 Wu X, Lee CC, Muzny DM, Caskey CT (1989) Urate oxidase: Primary structure and evolutionary implications. Proc Nati Acad Sci USA 86: 9412-9416 Wu X, Muzny DM, Lee CC, Caskey CT (1992) Two independent mutational events in the loss of urate oxidase, J Mol Evol 34: 78-84 Wu X, Wakamiya M, Vaishnav S, Geske R, Montgomery CM, Jr., Jones P, Bradley A et al., (1994) Hyperuricemia and urate nphropathy in urate oxidase-deficient mice. Proc Nati Acad Sci USA 91: 742-746 Zittoun R, Dauchy F, Teillaud C, Barthelemy M, Bouchard P (1976) Le traitement des hyperuricemies en hematologie par 1 'urate-oxydase et 1' allopurinol. Ann Med LIST OF SEQUENCES < 110 > HERSHFJELD, MICHAEL S. KELLY, SUSAN J. < 120 > URATO OXIDASE < 130 > 1579-379 < 140 > PCT / US99 / 17678 < 141 > 1999-08-05 < 160 > 11 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 915 < 212 > DNA < 213 > Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (915) < 220 > < 223 > Description of the Artificial Sequence: PBC Chimera < 400 > 1 atg gct cat tac cgt aat gac tac aaa aag aat gat gag gta gag ttt 48 Met Wing His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 gtc cga act ggc tat ggg aag gat atg ata aaa gtt etc cat att cag 96 Val Arg Thr Gly Tyr Gly Lys Asp Met lie Lys Val Leu His lie Gln 20 25 30 cga gat gga aaa tat falls age att aaa gag gtg gca act tea gtg caa 144 Arg Asp Gly Lys Tyr His Ser lie Lys Glu Val Wing Thr Ser Val Gln 35 40 45 ctg act ttg age tec aaa aaa gat tac ctg cat gga gac aat tea gat 192 Leu Thr Leu Be Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 gtc ate ect here gac acc ate a g aac a gt aat gtc ctg gcg aag 240 Val lie Pro Thr Asp Thr lie Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 ttc aaa ggc ate aaa age ata gaa act ttt gctg act ate tgt gag 288 Phe Lys Gly lie Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 cat ttc ctt tet tec ttc aag cat gtc ate aga gct ca gtc tat gtg 336 His Phe Leu Ser Ser Phe Lys His Val He Arg Ala Gln Val Tyr Val 100 105 110 gaa gaa gtt ect tgg aag cgt ttt gaa aag aat gga gtt aag cat gtc 384 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly V al Lys His Val 115 120 125 cat gca ttt att tat act ect act gga acg falls ttc tgt gag gtt gaa 432 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 cag ata agg aat gga ect cea gtc att cat tet gga ate aaa gac cta 480 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 aaa gtc ttg aaa here acc ggc ttt gag gga ttc ate aag gac 528 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 cag ttc acc acc ect ect gag gtg aag gac cgg tgc ttt gee acc to ca 576 Gln Phe Thr Thu Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 * 190 gtg tac tgc aaa tgg cgc tac falls cag ggc aga gat gtg gac ttt gag 624 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 gee acc tgg gac act gtt agg age att gtc ctg cag aaa ttt gct ggg 672 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Wing Gly 210 215 220 ecc tat gac aaa ggc gag tac tea ecc tet gtg cag aag acc etc tat 720 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 gat ate cag gtg etc tec ctg age cga gtt ect gag ata gaa gat atg 768 Asp He Gln Val Leu Ser Leu Ser Arg Val Pro Glu He Glu Asp Met 245 250 255 gaa ate age ctg cea aac att falls tac ttc aat ata gac atg tec aaa 816 Glu He Ser Leu Pro Asn lie His Tyr Phe Asn He Asp Met Ser Lys 260 265 270 atg ggt ctg ate aac aag gaa gtc ttg ctg cea tta gac aat cea 864 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 tat gga aaa att act ggt here gtc aag agg aag ttg tet tea aga ctg 912 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 tga 915 < 210 > 2 < 211 > 304 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: PBC Chimera < 400 > 2 Met Wing His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met He Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val He Pro Thr Asp Thr He Lys Asn Thr Val Asn Val Leu Wing Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val "He Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Wing Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Ser Leu Ser Arg Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Phe Asn As As Met Met Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 < 210 > 3 < 211 > 915 < 212 > DNA < 213 > Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (915) < 220 > < 223 > Description of the Artificial Sequence: Chimera PKS < 400 > 3 atg gct cat tac cgt aat gac tac aaaaag aat gat gag gta gag ttt 48 Met Wing His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 gtc cga act ggc tat ggg aag gat atg ata aaa gtt etc cat att cag 96 Val Arg Thr Gly Tyr Gly Lys Asp Met He Lys Val Leu His He Gln 20 25 30 cga gat gga aaa tat falls age att aaa * gag gtg gca act tea gtg caa 144 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Wing Thr Ser Val Gln 35 40 45 ctg act ttg age tec aaa aaa gat tac ctg cat gga gac aat tea gat 192 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 gtc ate ect here gac acc ate aag aac here gtt aat gtc ctg gcg aag 240 Val He Pro Thr Asp Thr He Lys Asn Thr Val Asn Val Leu Wing Lys 65 70 75 80 ttc aaa ggc ate aaa age ata gaa act ttt gct gtg act ate tgt gag 288 Phe Lys Gly He Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 cat ttc ctt tet tec ttc aag cat gtc ate aga gct ca gtc tat gtg 336 His Phe Leu Ser Ser Phe Lys His Val He Arg Ala Gln Val Tyr Val 100 105 110 gaa gaa gtt ect tgg aag cgt ttt gaa aag aat gga gtt aag cat gtc 384 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 cat gca ttt att tat act act act gga acg falls ttc tgt gag gtt gaa 432 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 cag ata agg aat gga ect cea gtc att cat tet gga ate aaa gac cta 480 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 aaa gtc ttg aaa here acc ggc ttt gag gga ttc ate aag gac 528 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 cag ttc acc acc ect ect gag gtg aag gac cgg tgc ttt gee acc to ca 576 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 gtg tac tgc aaa tgg cgc tac falls cag ggc aga gat gtg gac ttt gag 624 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 gee acc tgg gac act gtt agg age att gtc ctg cag aaa ttt gct ggg 672 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Wing Gly 210 215 220 ecc tat gac aaa ggc gag tac tcg tcg ecc tet gtc cag aag here etc tat 720 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 gac ate cag gtg etc acc ctg ggc cag gtt ect gag ata gaa gat atg 768 Asp He Gln Val Leu Thr Leu Gly Gln Val Pro Glu He Glu Asp Met 245 250 255 gaa ate age ctg cea aat att falls tac tta aac ata gac atg tec aaa 816 Glu He Ser Leu Pro Asn He His Tyr Leu Asn He Asp Met Ser Lys 260 265 270 atg gga ctg ate aac aag gaa ga gtc ttg cta ect tta gac aat cea 864 Met Gly Leu He Asn Lys Glu Glu Val <; Leu Leu Pro Leu Asp Asn Pro 275 280 285 tat gga aaa att act ggt here gtc aag agg aag ttg tet tea aga ctg 912 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 tga 915 < 210 > 4 < 211 > 304 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Chimera PKS < 400 > 4 Met Wing His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met He Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Ala Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val He Pro Thr Asp Thr He Lys Asn Thr Val Asn Val Leu Wing Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val He Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Ala Gly 210 215 220 < Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Thr Leu Gly Gln Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Leu Asn He Asp Met Ser Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 < 210 > 5 < 211 > 304 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Baboon D3H < 400 > 5 Met Wing His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Wing Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 He He Pro Thr Asp Thr He Lys Asn Thr Val His Val Leu Ala Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Wing Phe Gly Val Asn He Cys Glu 85 90 95 Tyr Phe Leu Ser Ser Phe Asn His Val He Arg Wing Gln Val Tyr Val 100 105 110 Glu Glu He Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Leu Arg Ser Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 < Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Gly Thr He Arg Asp Leu Val Leu Glu Lys Phe Wing Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Ser Leu Ser Arg Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Phe Asn As As Met Met Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 < 210 > 6 < 211 > 304 < 212 > PRT < 213 > baboon < 400 > 6 Met Wing Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Wing Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 He He Pro Thr Asp Thr He Lys Asn Thr Val His Val Leu Wing Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Wing Phe Gly Val Asn He Cys Glu 85 90 95 Tyr Phe Leu Ser Ser Phe Asn His Val He Arg Wing Gln Val Tyr Val 100 105 110 Glu Glu He Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln Leu Arg Ser Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 4 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Gly Thr He Arg Asp Leu Val Leu Glu Lys Phe Wing Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Ser Leu Ser Arg Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Phe Asn As As Met Met Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 300 < 210 > 7 < 211 > 304 < 212 > PRT < 213 > pig < 400 > 7 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met He Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Wing Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val He Pro Thr Asp Thr He Lys Asn Thr Val Asn Val Leu Wing Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val He Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Wing Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Thr Leu Gly Gln Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Leu Asn He Asp Met Ser Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Arg He Thr Gly Thr Val Lys Arg Lys Leu Thr Ser Arg Leu 290 295 300 < 210 > 8 < 211 > 298 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: truncated amino PBC < 400 > 8 sp Tyr Lys Lys Asn Asp Glu Val Glu Phe Val Arg Thr Gly Tyr Gly 1 5 10 15 Lys Asp Met He Lys Val Leu His He Gln Arg Asp Gly Lys Tyr His 20 25 30 Ser He Lys Glu Val Wing Thr Ser Val Gln Leu Thr Leu Ser Ser Lys 35 40 45 Lys Asp Tyr Leu His Gly Asp Asn Ser Asp Val He Pro Thr Asp Thr 50 55 60 He Lys Asn Thr Val Asn Val Leu Wing Lys Phe Lys Gly He Lys Ser 65 70 75 80 He Glu Thr Phe Wing Val Thr He Cys "Glu His Phe Leu Ser Ser Phe 85 90 95 Lys His Val He Arg Wing Gln Val Tyr Val Glu Glu Val Pro Trp Lys 100 105 110 Arg Phe Glu Lys Asn Gly Val Lys His Val His Wing Phe He Tyr Thr 115 120 125 Pro Thr Gly Thr His Phe Cys Glu Val Glu Gln He Arg Asn Gly Pro 130 135 140 Pro Val He His Ser Gly He Lys Asp Leu Lys Val Leu Lys Thr Thr 145 150 155 160 Gln Ser Gly Phe Glu Gly Phe He Lys Asp Gln Phe Thr Thr Leu Pro 165 170 175 Glu Val Lys Asp Arg Cys Phe Wing Thr Gln Val Tyr Cys Lys Trp Arg 180 185 190 Tyr His Gln Gly Arg Asp Val Asp Phe Glu Wing Thr Trp Asp Thr Val 195 200 205 Arg Ser He Val Leu Gln Lys Phe Wing Gly Pro Tyr Asp Lys Gly Glu 210 215 220 Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr Asp He Gln Val Leu Ser 225 230 235 240 Leu Ser Arg Val Pro Glu He Glu Asp Met Glu He Ser Leu Pro Asn 245 250 255 He His Tyr Phe Asn He Asp Met Being Lys Met Gly Leu He Asn Lys 260 265 270 Glu Glu Val Leu Leu Pro Leu Asp Asn Pro Tyr Gly Lys He Thr Gly 275 280 285 Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 < 210 > 9 < 211 > 301 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: truncated carboxy PBC < 400 > 9 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met He Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Wing Thr Ser Val Gln 35 40 * 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val He Pro Thr Asp Thr He Lys Asn Thr Val Asn Val Leu Ala Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val He Arg Ala Gln Val Tyr Val 100 105 110 Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Wing Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Ser Leu Ser Arg Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Phe Asn As As Met Met Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser 290 295 300 < 210 > 10 < 211 > 298 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Truncated carboxy PKS < 400 > 10 Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe Val Arg Thr Gly Tyr Gly 1 5 10 15 Lys Asp Met He Lys Val Leu His He Gln Arg Asp Gly Lys Tyr His 20 25 30 Ser He Lys Glu Val Wing Thr Ser Val Gln Leu Thr Leu Ser Ser Lys 35 40 45 Lys Asp Tyr Leu His Gly Asp Asn Ser Asp Val He Pro Thr Asp Thr 50 55 60 He Lys Asn Thr Val Asn Val Leu Wing Lys Phe Lys Gly He Lys Ser 65 70 75 80 He Glu Thr Phe Wing Val Thr He Cys Glu His Phe Leu Ser Ser Phe 85 90 95 Lys His Val He Arg Wing Gln Val Tyr Val Glu Val Val Glu Pro Lys 100 105 110 Arg Phe Glu Lys Asn Gly Val Lys His Val His Wing Phe He Tyr Thr 115 120 125 Pro Thr Gly Thr His Phe Cys Glu Val Glu Gln He Arg Asn Gly Pro 130 135 140 Pro Val He His Ser Gly He Lys Asp Leu Lys Val Leu Lys Thr Thr 145 150 155 160 Gln Ser Gly Phe Glu Gly Phe He Lys Asp Gln Phe Thr Thr Leu Pro 165 170 175 Glu Val Lys Asp Arg Cys Phe Wing Thr Gln Val Tyr Cys Lys Trp Arg 180 185 190 Tyr His Gln Gly Arg Asp Val Asp Phe Glu Wing Thr Trp Asp Thr Val 195 200 205 Arg Ser He Val Leu Gln Lys Phe Wing Gly Pro Tyr Asp Lys Gly Glu 210 215 220 Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr Asp He Gln Val Leu Thr 225 230 235 240 Leu Gly Gln Val Pro Glu He Glu Asp Met Glu He Ser Leu Pro Asn 245 250 255 He His Tyr Leu Asn He Asp Met Being Lys Met Gly Leu He Asn Lys 260 265 270 Glu Glu Val Leu Leu Pro Leu Asp Asn Pro Tyr Gly Lys He Thr Gly 275 280 285 Thr Val Lys Arg Lys Leu Ser Ser Arg Leu 290 295 < 210 > 11 «< 211 > 301 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Truncated carboxy PKS < 400 > 11 Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe 1 5 10 15 Val Arg Thr Gly Tyr Gly Lys Asp Met He Lys Val Leu His He Gln 20 25 30 Arg Asp Gly Lys Tyr His Ser He Lys Glu Val Wing Thr Ser Val Gln 35 40 45 Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp 50 55 60 Val He Pro Thr Asp Thr He Lys Asn Thr Val Asn Val Leu Wing Lys 65 70 75 80 Phe Lys Gly He Lys Ser He Glu Thr Phe Wing Val Thr He Cys Glu 85 90 95 His Phe Leu Ser Ser Phe Lys His Val He Arg Ala Gln Val Tyr Val 100 105 no Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val 115 120 125 His Wing Phe He Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu 130 135 140 Gln He Arg Asn Gly Pro Pro Val He His Ser Gly He Lys Asp Leu 145 150 155 160 Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe He Lys Asp 165 170 175 Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Wing Thr Gln 180 185 190 Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu 195 200 205 Wing Thr Trp Asp Thr Val Arg Ser He Val Leu Gln Lys Phe Wing Gly 210 215 220 Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr 225 230 235 240 Asp He Gln Val Leu Thr Leu Gly Gln Val Pro Glu He Glu Asp Met 245 250 255 Glu Be Ser Leu Pro Asn He His Tyr Leu Asn He Asp Met Ser Lys 260 265 270 Met Gly Leu He Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro 275 280 285 Tyr Gly Lys He Thr Gly Thr Val Lys Arg Lys Leu Ser 290 295 300

Claims

55 NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered as a novelty and therefore the contents of the following are declared as property: CLAIMS 1. A protein comprising a recombinant uricase protein of a mammalian species that has been modified to insert one or more lysine residues.
2. A protein according to claim 1, characterized in that the recombinant protein is a chimeric protein of two or more mammalian amino acid sequences.
3. A protein according to claim 2, characterized in that the recombinant uricase chimeric protein comprises 304 amino acids, the first N-terminal portion of 225 of said 304 amino acids are amino acids 1-225 of porcine uricase and the remaining amino acids of said 304 amino acids, with amino acids 226-304 of baboon uricase.
4. A protein according to claim 2, characterized in that the recombinant uricase chimeric protein comprises 304 amino acids, the first N-terminal portion of 228 of said 304 amino acids are amino acids 1-288 of porcine uricase and the remaining amino acids of said 304 amino acids are amino acids 289-304 56 of uricasa of baboon.
5. A recombinant uricase protein selected from the group consisting of SEQ ID NO: 2, 4, 8, 9, 10, and 11.
6. An isolated and purified nucleic acid molecule encoding the recombinant uricase in accordance with claim claim 1.
7. An isolated and purified nucleic acid molecule encoding the recombinant uricase according to claim 3.
8. An isolated and purified nucleic acid molecule encoding the recombinant uricase according to claim as claimed in claim 4.
9. An isolated and purified nucleic acid molecule encoding the recombinant uricase according to claim 5.
10. An isolated and purified nucleic acid molecule according to claim 9 wherein a base sequence of SEQ ID NO: 1.
11. An isolated and purified nucleic acid molecule according to claim claimed in the claim 9 having a base sequence of SEQ ID NO: 3.
12. A vector comprising a nucleic acid molecule according to claim 1.
13. A vector comprising an acid molecule. nucleic acid according to claim 9.
14. A host cell comprising a vector according to claim 12.
15. A host cell comprising a vector according to claim 13.
A method for increasing the non-deleterious PEG binding sites available in a uricase protein comprising mutating a uricase protein whereby at least one lysine residue is introduced therein.
17. A method for increasing the non-deleterious PEG binding sites available in a uricase protein comprising mutating an uricase protein whereby at least one lysine residue is introduced therein at the site of an arginine.