WO2008039690A2 - Recombinant renalase - Google Patents

Abstract

The present invention provides methods of producing recombinant renalase using a methylotrophic yeast such as Pichia pastoris. The invention provides a method for producing recombinant renalase protein by culturing a Pichia pastoris harboring a renalase coding sequence operably linked to a promoter. The recombinant Pichia pastoris is cultured under conditions that permit or induce expression of the renalase protein. The recombinant renalase protein may then be recovered from cell extracts, from culture medium or culture supernatants. Recombinant renalase produced in accordance with the invention is stable, and abundant amounts of renalase can be produced. The present invention also provides for recombinant forms of renalase, including recombinant vectors and cells containing a renalase coding sequence.

Description

RECOMBINANT RENALASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application No. 60/846,760 filed September 25, 2006, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was supported in part by funds obtained from the U.S. Government (Veteran Administration (Awards: Merit Review and Career Development) and the National Institutes of Health (Grant Numbers: DK48105B, K08DK02917, and DK064317), and the U.S. Government may therefore have certain rights in the invention.

FIELD OF THE INVENTION

[0003] This invention relates generally to mammalian Monoamine Oxidase C (MAO-C), also known as renalase, recombinant forms of renalase, and to methods of producing recombinant renalase.

BACKGROUND OF THE INVENTION

[0004] Several large studies demonstrate that chronic kidney disease (CKD) is associated with a marked increase in cardiovascular complications. Approximately 400,000 Americans have end-stage renal disease (ESRD), and over 300,000 of these patients require maintenance dialysis. The mortality rate remains above 20 percent per year with the use of dialysis, with more than half of the deaths related to cardiovascular disease. Cardiovascular complications are also increased in patients with CKD who are not on dialysis.

[0005] Although the reasons for this are not entirely clear, it is generally believed that in end stage renal disease dialysis procedure fails to replicate important functions of the natural organ. In addition to maintaining fluid and electrolyte homeostasis, the kidney also serves as an endocrine organ. For example, the kidney is the main source of erythropoietin and renin, and is involved in the synthesis of Vitamin D and in the metabolism of the antioxidant glutathione. Perhaps our incomplete understanding of the endocrine function of the kidney rney oc e o. -

results in less than adequate therapeutic regimens and contributes to the higher cardiovascular morbidity and mortality observed in chronic kidney disease.

SUMMARY OF THE INVENTION

[0006] While searching for novel proteins secreted by the kidney, we identified a flavin adenine dinucleotide-dependent amine oxidase (renalase) that is secreted into the blood by the kidney and metabolizes circulating catecholamines (Xu et al. J Clin Invest. 2; 1 15(5): 1275- 1280 (2005) and WO 2005/089505, each of which is specifically incorporated by reference herein in its entirety). The plasma concentration of renalase is markedly reduced in patients with end-stage renal disease, as compared with healthy subjects. Renalase infusion in rats caused a decrease in cardiac contractility, heart rate, and blood pressure and prevented a compensatory increase in peripheral vascular tone. These results identify renalase as a novel amine oxidase that is secreted by the kidney, circulates in blood, and modulates cardiac function and systemic blood pressure.

[0007] It is well documented that patients with renal failure have a heightened level of circulating catecholamines (i.e. norepinephrine) and that such changes are associated with higher cardiovascular mortality. Based on our data we hypothesize that the loss of renal function leads to a reduction in blood renalase levels, which contributes to the increase in circulating catecholamine levels and cardiovascular risks.

[0008] Renalase is therefore an attractive therapeutic candidate for patients with, inter alia, end stage renal disease.

[0009] The present invention provides methods of producing recombinant renalase using a methylotrophic yeast, including those of the genera Hansenula, Pichia, Candida, and Torulopsis, and including such species as Pichia pastor is. For example, the invention provides a method for producing recombinant renalase protein by culturing a methylotrophic yeast, such as a Pichia past oris, harboring a renalase coding sequence operably linked to a promoter. The recombinant yeast is cultured under conditions that permit or induce expression of the renalase protein, as disclosed herein. The recombinant renalase protein may then be recovered from cell extracts or from culture medium or culture supernatants. The renalase protein produced according to the method may be a mammalian renalase protein, such as a human renalase protein.

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[0010] In certain embodiments, and as described herein, the expression of the renalase protein in, for example, Pichia pastoris may be induced by the addition of methanol to the culture medium.

[0011] Where the recombinant renalase is recovered from the culture medium, the renalase protein may be secreted from the cultured cells via a Pichia alpha factor signal sequence, or may be secreted via the native renalase signal sequence.

[0012] As disclosed herein, recombinant renalase produced in yeast systems, such as Pichia pastoris, is stable, that is the protein is not proteolytically degraded, and abundant amounts of renalase can be produced. For example, a recombinant Pichia pastoris as described herein may be cultured for eight days or more, and during this culturing period the recombinant Pichia pastoris continues to produce and secrete recombinant renalase into the culture medium.

[0013] Another aspect of the invention provides a recombinant human renalase truncated at the N-terminus by three amino acids, for example, a human renalase consisting of amino acids 4-342 of SEQ ID NO:2, or a similarly truncated form of a renalase isoform described herein. This truncated form may be conveniently produced by cloning the renalase gene in frame with the alpha factor signal sequence and adjacent to a Kex2 cleavage signal, as described herein.

[0014] In another aspect, the invention provides a recombinant methylotrophic yeast, such as Pichia pastoris, harboring a mammalian renalase gene, cDNA or coding sequence. The recombinant cells of the invention, in certain embodiments, secrete the renalase protein, either via the native renalase signal sequence or via a P, pastoris alpha factor signal sequence.

[0015] In yet another aspect, the invention provides a recombinant vector, such as an expression or cloning vector, comprising a renalase gene, where the vector is suitable for expression, replication, or integration in Pichia pastoris. For example, a polynucleotide encoding renalase may be operably linked to a promoter and/or termination signal suitable for permitting or controlling expression in Pichia pastoris.

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BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. Renalase secretion by Pichia Pastoris. X-33 clones are transformed with pPICZ_ren_ 1 -342, and renalase synthesis was induced by methanol at 0 hrs. Supernatant collected was at indicated times (72 hrs and up to 8 days), and renalase expression was assessed by western blotting using a c-myc antibody. The letter P indicates a positive control for the c-myc antibody.

[0017] Figure 2. Renalase secretion by Pichia Pastoris. X-33 clones and KM71H were transformed with pPICZα _ren_4-342 and renalase synthesis was induced by methanol at 0 hrs. Supernatant was collected at indicated times, and renalase expression was assessed by western blotting using a c-myc antibody. The letter P indicates a positive control for the c- myc antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0018] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by reference.

[0019] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] Definitions

[0022] As used herein, each of the following terms has the meaning associated with it in this section.

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[0023] The articles "a" and "an" are used herein to refer to one or to more than one (i.e.. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0024] As used herein, the term "adjacent" is used to refer to nucleotide sequences which are directly attached to one another, having no intervening nucleotides. By way of example, the pentanucleotide 5'-AAAAA-3' is adjacent the trinucleotide 5'-TTT-3' when the two are connected thus: 5'-AAAAATTT-3' or 5'-TTTAAAAA-3', but not when the two are connected thus: 5'-AAAAACTTT-3\

[0025] As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:

Full Name Three-Letter Co oddee One-Letter Code

Aspartic Acid Asp D

Glutamic Acid GIu E

Lysine Lys K

Arginine Arg R

Histidine His H

Tyrosine Tyr Y

Cysteine Cys C

Asparagine Asn N

Glutamine GIn Q

Serine Ser S

Threonine Thr T

Glycine GIy G

Alanine Ala A

Valine VaI V

Leucine Leu L

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Isoleucine He I

Methionine Met M

Proline Pro P

Phenylalanine Phe F

Tryptophan Trp W

[0026] A "coding region" or "coding sequence" of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.

[0027] A "coding region" or "coding sequence" of an mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues corresponding to amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0028] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e.. rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0029] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0030] "Expression vector" refers to a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression

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vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.

[0031] A first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent one another or if the two regions are separated by no more than about 1000 nucleotide residues, and preferably no more than about 100 nucleotide residues.

[0032] As used herein, the term "fragment" as applied to a polynucleotide or gene refers to a segment or portion of a reference full length polynucleotide molecule or gene, wherein the fragment is less than the full length, An example of a fragment nucleic acid sequence or molecule is a segment or portion of the full length renalase cDNA sequence or molecule, respectively. Another example of a fragment nucleic acid sequence or molecule is a segment or portion of a full length genomic renalase DNA sequence or molecule, respectively. A fragment may be any length that is less than the full length of the natural or native cDNA or gene. Examples of fragments include nucleic acids of at least about 20 nucleotides in length, at least about 50 nucleotides, at least about 50 to about 100 nucleotides, at least about 100 to about 200 nucleotides, at least about 200 nucleotides to about 300 nucleotides, at least about 300 to about 350, at least about 350 nucleotides to about 500 nucleotides, at least about 500 to about 600, at least about 600 nucleotides to about 650 nucleotides, at least about 650 to about 800, or at least 800 to about 1000 nucleotides in length.

[0033] "Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3ΑTTGCC5' and 3'TATGGC share 50% homology.

[0034] As used herein, "homology" is used synonymously with "identity."

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[0035] In addition, when the terms "homology" or "identity" are used herein to refer to the nucleic acids and proteins, it should be construed to be applied to homology or identity at both the nucleic acid and the amino acid sequence levels. A first oligonucleotide anneals with a second oligonucleotide with "high stringency" or "under high stringency conditions" if the two oligonucleotides anneal under conditions whereby only oligonucleotides which are at least about 60%, more preferably at least about 65%, even more preferably at least about 70%, yet more preferably at least about 80%, and preferably at least about 90% or, more preferably, at least about 95% complementary anneal with one another. The stringency of conditions used to anneal two oligonucleotides is a function of, among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the oligonucleotides, the G-C content of the oligonucleotides, and the expected degree of non- homology between the two oligonucleotides, if known. Methods of adjusting the stringency of annealing conditions are known (see, e.g., Sambrook et al., 1989, In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0036] The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990, J. MoI. Biol. 215:403- 410), and can be accessed, for example, at the BLAST site of the National Center for Biotechnology Information (NCBI) world wide web site at the National Library of Medicine (NLM) at the National Institutes of Health (NIH). BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" at the NCBI web site), using the following parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1 ; expectation value 10.0; and word size = 1 1 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.

[0037] To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).

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Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used as available on the website of the National Center for Biotechnology Information of the National Library of Medicine at the National Institutes of Health.

[0038] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

[0039] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. Such natural allelic variations can typically result in 1 -5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.

[0040] Moreover, nucleic acid molecules encoding proteins of the invention from other species (homologs), which have a nucleotide sequence which differs from that of the renalase species described herein are within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologs of a cDNA of the invention can be isolated based on their identity to, for example, human renalase, using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.

[0041] An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or

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proteins. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0042] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0043] By describing two polynucleotides as "operably linked" is meant that a single- stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region. Preferably, when the nucleic acid encoding the desired protein further comprises a promoter/regulatory sequence, the promoter/regulatory is positioned at the 5' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell. Together, the nucleic acid encoding the desired protein and its promoter/regulatory sequence comprise a "transgene." Two polypeptides do not necessarily need to be adjacent to each other in order to be operably linked.

[0044] As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.

[0045] A "polyadenylation sequence" is a polynucleotide sequence which directs the addition of a poly A tail onto a transcribed messenger RNA sequence.

[0046] A "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.

[0047] The term "nucleic acid" typically refers to large polynucleotides.

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[0048] Conventional notation is used herein to describe polynucleotide sequences: the left- hand end of a single-stranded polynucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.

[0049] The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences."

[0050] A "portion" of a polynucleotide means at least at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.

[0051] "Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

[0052] "Probe" refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

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[0053] "Recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

[0054] A "recombinant polypeptide" is one which is produced upon expression of a recombinant polynucleotide.

[0055] As used herein, "renalase" is a molecule that has high and/or significant sequence identity with the renalase polypeptides disclosed herein. Generally, a renalase coding sequence will share at least about 40% sequence identity with a nucleic acid having the sequence SEQ ID NO: 1 or a nucleic acid sequence encoding a renalase isoform disclosed herein. More preferably, a nucleic acid encoding a renalase has at least about 45% identity, or at least about 50% identity, or at least about 55% identity, or at least about 60% identity, or at least about 65% identity, or at least about 70% identity, or at least about 75% identity, or at least about 80% identity, or at least about 85% identity, or at least about 90% identity, or at least about 95% identity, or at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 1 or a nucleic acid sequence encoding a renalase isoform disclosed herein. Even more preferably, the nucleic acid encoding renalase is SEQ ID NO: 1. In nature, renalase is a monoamine oxidase secreted by the kidney, and/or other cells, and metabolizes biogenic monoamines such as dopamine, norepinephrine, and epinephrine.

[0056] Thus term ''renalase" also includes renalase isoforms. The renalase gene contains 9 exons spanning 310, 188 bp in chromosome 10 of the human genome. The renalase clone (SEQ ID NO: 1, GenBank accession number: BC005364) is the gene containing exons 1 , 2, 3, 4, 5, 6. 8. There are at least two additional alternatively-spliced forms of renalase protein as shown in the human genome database. One alternatively spliced form contains exons 1, 2, 3, 4, 5, 6, 9, identified by clones in the human genome database as GenBank accession number AK002080 (SEQ ID NO: 3) and NMJ) 18363 (SEQ ID NO: 4). The other alternatively spliced form contains exons 5, 6, 7, 8, identified by clones in the human genome database as GenBank accession number BX648154 (SEQ ID NO: 5). In summary, there is one renalase gene with at least 4 different transcripts (i.e., 948 bp, 1447 bp, 1029 bp, and 3385 bp) encoding 2 different renalase isoforms (342 aa and 315 aa). In addition, a nucleotide polymorphism that results in an amino acid change from 37E to 37D has been

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identified. This polymorphism has been found at the 37th amino acid in each of the two iso forms. A survey of the prevalence of the polymorphism in about 120 subjects has determined the change exists in approximately 35% of the subjects.

[0057] As used herein, the term "active renalase" refers to a renalase protein that can metabolize catecholamines in vitro, and that has measurable hemodynamic effects on blood pressure, heart rate, cardiac contractility etc, in vivo.

[0058] A "restriction site" is a portion of a double-stranded nucleic acid which is recognized by a restriction endonuclease. A portion of a double-stranded nucleic acid is "recognized" by a restriction endonuclease if the endonuclease is capable of cleaving both strands of the nucleic acid at the portion when the nucleic acid and the endonuclease are contacted.

[0059] A first oligonucleotide anneals with a second oligonucleotide "with high stringency" if the two oligonucleotides anneal under conditions whereby only oligonucleotides which are at least about 70%, or at least about 73%, more preferably, at least about 75%, even more preferably, at least about 80%, even more preferably, at least about 85%, yet more preferably, at least about 90%, and most preferably, at least about 95%, complementary anneal with one another. The stringency of conditions used to anneal two oligonucleotides is a function of, among other factors, temperature, ionic strength of the annealing medium, the incubation period, the length of the oligonucleotides, the G-C content of the oligonucleotides, and the expected degree of non-homology between the two oligonucleotides, if known. Methods of adjusting the stringency of annealing conditions are known (see, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0060] By the term "'exogenous nucleic acid" is meant that the nucleic acid has been introduced into a cell using technology which has been developed for the purpose of facilitating the introduction of a nucleic acid into a cell.

[0061] By "tag" polypeptide is meant any protein which, when linked by a peptide bond to a protein of interest, may be used to localize the protein, to purify it from a cell extract, to immobilize it for use in binding assays, or to otherwise study its biological properties and/or function.

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[0062] Description

[0063] The present invention provides a method for producing recombinant renalase protein. The method involves culturing a methylotrophic yeast, such as of the genus Pichia (i.e., Pichia past oris), harboring a recombinant renalase gene operably linked to one or more regulatory sequences, and under conditions that permit expression of the renalase protein. The renalase protein may then be recovered from the recombinant cell, or from the culture media or supernatants.

[0064] In certain embodiments, the method of the present invention allows for secretion of recombinant renalase into the culture medium at high levels, providing extremely high yields of active renalase that may be purified and processed for numerous therapeutic applications, including the treatment of end-stage renal disease.

[0065] To express the renalase gene, the coding sequence is operably linked to a 5' regulatory region and a 3' termination sequence in an expression cassette, which may be inserted into the host microorganism. The spacing between the renalase coding sequence and the regulatory elements, as well as the spacing of the regulatory elements themselves, can be optimized for improved expression of the recombinant protein. Exemplary regulatory regions include the AOXl or DASl 5' regulatory regions as described in U.S. Patent 5,707,828, which is hereby incorporated by reference. These or other appropriate regulatory regions for expression in P. pastoris are linked at their 3' end of the sequence to the ATG start codon of the structural gene. i.e. a renalase gene.

[0066] Several regulatory regions have been characterized and can be employed in conjunction with the expression of renalase in Pichia pastoris. Exemplary 5' regulatory regions are the primary alcohol oxidase (AOXl), dihydroxyacetone synthase (DASl), and the p40 regulatory regions, derived from Pichia pastoris and the like. In certain embodiments, the 5' regulatory region responds to methanol-containing media, such regulatory regions such as AOXl and DAS l , are disclosed in U.S. Patent 4,855,231, which is incorporated herein by reference.

[0067] 3' termination sequences may function to terminate, polyadenylate and/or stabilize the messenger RNA encoded by the structural gene when operably linked to the gene. However, the particular 3' termination sequence is not believed to be critical to the practice of the present invention. Exemplary 3' termination sequences for the practice of this invention

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include but are not limited to those derived from Pichia pastoris, such as those selected from the group consisting of the 3' termination sequences of AOXl gene, DAS l gene, p40 gene, and HIS4 gene.

[0068] Pichia pastoris may be transformed with a variety of renalase coding sequences, including full-length renalase coding sequences and fragments thereof. Renalase genes, renalase cDNA, and the isolation thereof, are described in WO 2005/089505, which is incorporated herein by reference. Following the isolation of a renalase gene or cDNA, the coding sequence is inserted into a suitable Pichia pastoris vector such as a plasrnid or linear site-specific integrative vector.

[0069] Plasmid type vectors are often employed in recombinant DNA technology. Plasmids are circular extra-chromosomal double-stranded DNA found in microorganisms. Plasmids have been found to occur in single or multiple copies per cell. Plasmid DNA possesses information required for plasmid reproduction, e.g. an autonomous replication sequence such as those disclosed in U.S. Patent 4,837, 148, which is hereby incorporated by reference. The autonomous replication sequences provide a suitable means for maintaining plasmids in Pichia pastoris. Additionally, the plasmid may also contain or encode one or more means of phenotypically selecting the plasmid in transformed cells.

[0070] Suitable integrative vectors for the practice of the present invention are the linear site-specific integrative vectors described in U.S. Patent No. 4,882,279, which is hereby incorporated by reference. These vectors comprise a serially arranged sequence of at least 1) a first insertable DNA fragment; 2) a selectable marker gene; and 3) a second insertable DNA fragment. An expression cassette containing a heterologous structural gene is inserted in this vector between the first and second insertable DNA fragments either before or after the marker gene. Alternatively, an expression cassette can be formed in situ if a regulatory region or promoter is contained within one of the insertable fragments to which the structural gene may be operably linked.

[0071] The first and second insertable DNA fragments are each at least about 200 nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the species to be transformed. The various components of the integrative vector are serially arranged forming a linear fragment of DNA such that the expression cassette and the selectable marker gene are positioned between the 3' end of the first insertable DNA fragment

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and the 5' end of the second insertable DNA fragment. The first and second insertable DNA fragments are oriented with respect to one another in the serially arranged linear fragment as they are oriented in the parent genome.

[0072] Nucleotide sequences useful as the first and second insertable DNA fragments are nucleotide sequences which are homologous with separate portions of the native genomic site at which genomic modification is to occur. For example, if genomic modification is to occur at the locus of the alcohol oxidase gene, the first and second insertable DNA fragments employed would be homologous to separate portions of the alcohol oxidase gene locus. Examples of nucleotide sequences which could be used as first and second insertable DNA fragments are deoxyribonucleotide sequences selected from the group consisting of the Pichiapastoris alcohol oxidase (AOX l) gene, dihydroxyacetone synthase (DASl ) gene, p40 gene and HIS4 gene. The AOXl gene, DAS l gene, p40 gene and HIS4 genes are disclosed in U.S. Patent Nos. 4,855,231 and 4,885,242, which are both incorporated herein by reference. The designation DASl is equivalent to the DAS designation originally used in U.S. Patents 4,855,231 and 4,885,242.

[0073] The first insertable DNA fragment may contain an operable regulatory region which may comprise the regulatory region utilized in the expression cassette. If the first insertable DNA fragment does not contain a regulatory region, a suitable regulatory region will need to be inserted, linked to the structural gene, in order to provide an operable expression cassette. Similarly, if no 3' termination sequence is provided at the insertion site to complete the expression cassette, a 3' termination sequence can be operably linked to the 3' end of the structural gene.

[0074] It is also highly desirable to include at least one selectable marker gene in the DNA used to transform the host strain. This facilitates selection and isolation of those organisms which have incorporated the transforming DNA. The marker gene confers a phenotypic trait to the transformed organism which the host did not previously possess, e.g., restoration of the ability to produce a specific amino acid where the untransformed host strain has a defect in the specific amino acid biosynthetic pathway, or provides resistance to antibiotics and the like. Exemplary selectable marker genes include the HIS4 gene (disclosed in U.S. Pat. No. 4,885,242, which is hereby incorporated by reference) and the ARG4 gene (disclosed in U.S. Pat. No. 4,818,700, which is incorporated herein by reference) from Pichiapastoris and Saccharomyces cerevisiae, the invertase gene (SUC2) (disclosed in U.S. Pat. No. 4,857,467,

54883 v I/DC 1 6 ttorney oc et o. -

which is incorporated herein by reference) from Saccharomyces cerevisiae, or the G418^R /kanamycin resistance gene from the E. coli transposable elements Tn601 or Tn903.

[0075] Those skilled in the art recognize that additional DNA sequences can also be incorporated into the vectors employed in the practice of the present invention, such as, for example, bacterial plasmid DNA, bacteriophage DNA, and the like. Such sequences enable the amplification and maintenance of these vectors in bacterial hosts.

[0076] The insertion of the renalase structural gene into suitable vectors may be accomplished by any suitable technique which cleaves the chosen vector at an appropriate site or sites and results in at least one operable expression cassette containing the renalase structural gene being present in the vector. Ligation of the renalase structural gene may be accomplished by any appropriate ligation technique such as utilizing T4DNA ligase.

[0077] The initial selection, propagation, and optional amplification of the ligation mixture of the renalase structural gene and a vector is preferably performed by transforming the mixture into a bacterial host such as E. coli (although the ligation mixture could be transformed directly into a yeast host). Suitable transformation techniques for E. coli are well known in the art. Additionally, selection markers and bacterial origins of replication necessary for the maintenance of a vector in a bacterial host are also well known in the art. The isolation and/or purification of the desired plasmid containing the renalase structural gene in an expression system may be accomplished by any suitable means for the separation of plasmid DNA from the host DNA. Similarly, the vectors formed by ligation may be tested, preferably after propagation, to verify the presence of the renalase gene and its operable linkage to a regulatory region and a 3' termination sequence. This may be accomplished by a variety of techniques including but not limited to endonuclease digestion, gel electrophoresis, or Southern hybridization.

[0078] Transformation of plasmids or linear vectors into yeast hosts may be accomplished by suitable transformation techniques including but not limited to those disclosed in U.S. Pat. No. 4,929,555 and U.S. Pat. No. 4,879,231, which are hereby incorporated by reference. Certain embodiments of the invention employ linear vectors, and select for insertions by Southern hybridization. Further, the Pichia pastoris may be selected for multiple integrations of a recombinant renalase gene, to further optimize expression and yield of the recombinant renalase.

54883 vl /DC 17 . -

[0079] The yeast host for transformation is a suitable methylotrophic yeast, such as of the genus Pichia (e.g. Pichia pastoris KM71). Suitable hosts also include the auxotrophic Pichia pastoris GSl 15 (NRRL Y- 15851); Pichia pastoris GS 190 (NRRL Y- 18014) disclosed in U.S. Pat. No. 4,818,700 (which is hereby incorporated by reference); and Pichia pastoris PPFl (NRRL Y- 18017), which is disclosed in U.S. Pat. No. 4,812,405, which is hereby incorporated by reference. Auxotrophic Pichia pastoris strains are advantageous for their ease of selection. Wild-type Pichia pastoris strains (such as NRRL Y-1 1430 and NRRL Y- 1 1431) may also be employed with equal success if a suitable transforming marker gene is selected, such as SUC2, or an antibiotic resistance such as resistance to G418. Other species of the genus Pichia that may be used in accordance with the present invention include Pichia ciferrii (as disclosed in U.S. Patent 6,638,735, which is hereby incorporated by reference) and Pichia methanolica.

[0080] Transformed Pichia cells can be selected for by using appropriate techniques including but not limited to culturing previously auxotrophic cells after transformation in the absence of the biochemical product required (due to the cell's auxotrophy), selection for and detection of a new phenotype ("methanol slow"), or culturing in the presence of an antibiotic which is toxic to the yeast in the absence of a resistance gene contained in the transformant.

[0081] Isolated transformed Pichia cells are cultured by appropriate fermentation techniques such as shake flask fermentation, high density fermentation or the technique disclosed by Cregg et al. in, High-Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris 5 Bio/Technology 479 (1987). Isolates may be screened by assaying for renalase production to identify those isolates with the highest renalase production level.

[0082] Transformed strains, which are of the desired phenotype and genotype, are grown in fermentors. For the large-scale production of recombinant DNA-based products in methylotrophic yeast, a three stage, high cell-density, batch fermentation system may be employed. When using a batch fermentation system, the yeast may be grown for up to 8 days, or more than 8 days is desirable. In the first, or growth stage, expression hosts are cultured in defined minimal medium with an excess of a non-inducing carbon source (e.g. glycerol). When grown on such carbon sources, heterologous gene expression is completely repressed, which allows the generation of cell mass in the absence of heterologous protein expression. During this growth stage, the pH of the medium is generally be maintained at

54883 vl /DC 18 . , -

about 5. Next, a short period of non-inducing carbon source limitation growth is allowed to further increase cell mass and derepress the methanol responsive promoter. The pH of the medium during this limitation growth period is adjusted to the pH value to be maintained during the production phase, which is typically at about pH 5 to about pH 6. Subsequent to the period of growth under limiting conditions, methanol alone ("limited methanol fed-batch mode") or a limiting amount of non-inducing carbon source plus methanol ("'mixed-feed fed- batch mode") are added in the fermentor, inducing the expression of the heterologous gene driven by a methanol responsive promoter. This third stage is the so-called production stage.

[0083] The present invention may be used to express any renalase protein or fragment thereof, including mammalian renalase proteins, such as a human renalase protein.

[0084] The renalase protein may be recovered from the cells, but is preferably recovered from the culture media or culture supernatants as secreted protein. The renalase structural gene, for example, the renalase cDNA, may be cloned in frame with a P, pastoris alpha factor signal sequence, allowing the renalase protein or truncated form, or fragment, to be secreted into the culture medium. The renalase gene may also be positioned such that the alpha factor signal sequence is cleaved upon secretion by the cells, such as by cloning the renalase gene in frame with the alpha factor signal sequence. In one embodiment, the renalase gene beginning with amino acid 4 of human renalase is cloned in frame with the alpha factor signal sequence and Kex2 cleavage signal, as described herein. In this embodiment, the recombinant human renalase is recovered from the culture medium, providing a renalase protein truncated by about 1 , 2, 3, or 4 amino acids. The renalase protein prepared by this method retains an intact FAD-binding domain.

[0085] Alternatively, the full length renalase protein may be cloned in frame with the appropriate regulatory sequences, so as to be secreted into the culture medium via the native renalase signal sequence.

[0086] The present invention further provides recombinant renalase proteins produced according to the methods of the invention. The present invention provides recombinant human renalase consisting of amino acids 4-342 of SEQ ID NO:2, for example, or other similarly truncated renalase proteins containing an intact FAD-binding site (such as similarly truncated renalase isoforms disclosed herein), but which are conveniently cloned to allow for efficient production in Pichia pastoris and efficient secretion into the culture media.

54883 vl /DC 19 orney oc e o. Y ALU-ΠU/U I WU

[0087] The invention further provides recombinant cells harboring a renalase gene, such as yeast recombinant cells, such as Pichia pastoris or Saccharomyces cerevisiae, and bacterial recombinant cells such as E. coli. The renalase gene may be contained on an expression or cloning plasmid, or integrated into the host chromosome. In certain embodiments of the invention, the recombinant cell is a Pichia pastoris that secretes a renalase protein, such as a full-length human renalase protein having a native renalase signal sequence. Alternatively, the recombinant cell of the invention secretes a renalase protein via cleavage of a P. pastoris alpha factor signal sequence, and in these embodiments, the recombinant renalase protein may be truncated, such as by one, two, three, or four amino acids. The truncated renalase protein of the invention generally has an intact, or functional, FAD-binding domain.

[0088] In another aspect, the present invention provides recombinant vectors suitable for expression in a Pichia pastoris. The recombinant vectors of the invention comprise regulatory sequences operably linked, and directing or controlling the expression of, a renalase coding sequence. In certain embodiment, the renalase coding sequence is cloned in frame with a P. pastoris alpha factor signal sequence, and/or the renalase coding sequence encodes the native renalase signal sequence.

[0089] Various expression constructs and kits for expressing the renalase protein in accordance with the invention are commercially available, such as from Invitrogen.

[0090] For producing recombinant renalase in accordance with the methods of the invention, the present invention utilizes isolated nucleic acids encoding renalase, or a fragment thereof, wherein the nucleic acid shares at least about 40% identity with the sequence of SEQ ID NO: 1 or a sequence encoding a renalase isoform disclosed herein. Alternatively, the nucleic acid is at least about 45% homologous, or at least about 50% homologous, or at least about 55% homologous, or at least about 60% homologous, or at least about 65% homologous, or at least about 70% homologous, or at least about 75% homologous, or at least about 80% homologous, or at least about 85% homologous, or at least about 90% homologous, or at least about 95% homologous, or at least about 98% homologous, or at least about 99% homologous to SEQ ID NO: 1 or a sequence encoding a renalase isoform.

[0091] Preferably, the protein encoded by the isolated nucleic acid encoding renalase is at least about 15% homologous, or at least about 20% homologous, or at least about 25% homologous, or at least about 30% homologous, or at least about 35% homologous, or at least

54883 vl/DC 20 . -

about 40% homologous, or at least about 45% homologous, or at least about 50% homologous, or at least about 55% homologous, or at least about 60% homologous, or at least about 65% homologous, or at least about 70% homologous, or at least about 75% homologous, or at least about 80% homologous, or at least about 85% homologous, or at least about 90% homologous, or at least about 95% homologous, or at least about 98%, or at least about 99% homologous to SEQ ID NO: 2 or a renalase isoform disclosed herein. A nucleic acid of SEQ ID NO: 1 can be translated to produce a human renalase protein comprising 342 amino acids with a calculated molecule mass of 37.8 kDa.

[0092] The present invention should not be construed as being limited solely to methods of using the specific renalase nucleic and amino acid sequences disclosed herein. It is readily apparent to one skilled in the art that other nucleic acids encoding renalase polypeptides such as those present in other species of mammals (e.g., ape, gibbon, bovine, ovine, equine, porcine, canine, feline, murine, and the like) can be used in accordance with the methods of the invention.

[0093] Further, any number of procedures may be used for the generation of mutant, derivative or variant forms of renalase using recombinant DNA methodology well known in the art such as, for example, that described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New York).

[0094] Procedures for the introduction of amino acid changes in a protein or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in Sambrook et al. (1989, supra); Ausubel et al. (1997, supra).

[0095] The invention further includes methods of expressing a nucleic acid encoding a mammalian renalase wherein a nucleic acid encoding a tag polypeptide is covalently linked thereto. That is, the invention encompasses expression of a chimeric nucleic acid wherein the nucleic acid sequences encoding a tag polypeptide is covalently linked to the nucleic acid encoding at least one renalase protein. Such tag polypeptides are well known in the art and include, for instance, green fluorescent protein (GFP), an influenza virus hemagglutinin tag polypeptide, myc, myc-pyruvate kinase (myc-PK), His6, maltose biding protein (MBP), a FLAG tag polypeptide, a HA tag polypeptide, and a glutathione-S-transferase (GST) tag polypeptide. However, the invention should in no way be construed to be limited to using the

54883 vl/DC 21 ttorney oc et o. A -

nucleic acids encoding the above-listed tag polypeptides. Rather, any nucleic acid sequence encoding a polypeptide which may function in a manner substantially similar to these tag polypeptides should be construed to be included as being useful in the present invention.

[0096] The nucleic acid comprising a nucleic acid encoding a tag polypeptide can be used to localize renalase within a cell, a tissue (e.g., a blood vessel, bone, and the like), and/or a whole organism (e.g., an amphibian and/or a mammalian embryo, and the like), detect renalase if secreted from a cell, and to study the role(s) of renalase in a cell. Further, addition of a tag polypeptide facilitates isolation and purification of the "tagged" protein such that the proteins of the invention can be produced and purified readily. For example, a tag polypeptide allows for easy purification from culture media in accordance with the present invention, and in certain embodiments, the tag may be proteolytically cleaved to obtain full- length renalase proteins, or desired truncations thereof.

[0097] The methods of the invention provide for recombinant forms of renalase, as well as analogs thereof. Analogs may differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

[0098] Other modifications (which do not normally alter primary sequence) include in vivo, or in vitro, chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps;

54883 vl /DC 22 ttorney oc et o. Y ALb- 1 1 υ/υ i wυ

e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0099] Also included are methods of expressing polypeptides which have been modified using ordinary molecular biological techniques, so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

[00100] The methods of the present invention should also be construed to encompass "mutants," "derivatives," and "variants" of the peptides of the invention (or of the DNA encoding the same) which mutants, derivatives and variants are renalase peptides which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or more base pairs) such that the resulting peptide (or DNA) is not identical to the sequences recited herein, but has the same biological property as the peptides disclosed herein, in that the peptide has biological/biochemical properties of the renalase peptide of the present invention.

[00101] Further, the invention should be construed to include methods of expressing renalase iso forms, and naturally occurring variants or recombinantly derived mutants of renalase sequences, which variants or mutants render the protein encoded thereby either more, less, or just as biologically active as the full-length clones of renalase.

[00102] Purification of the polypeptides including fragments, homologous polypeptides, muteins, analogs, derivatives and fusion proteins is well-known and within the skill of one having ordinary skill in the art. See, e.g., Scopes, Protein Purification, 2d ed. (1987). Accordingly, the present invention provides a method of purifying a renalase polypeptide from culture media or cellular extracts. Purification of renalase may be conducted by any protein purification known in the art, including but are not limited to, procedures of ion exchange chromatography, adsorption chromatography, ligand-bound affinity chromatography and gel permeation chromatography, solely or in combination.

54883 vl/DC 23 . -

[00103] The recombinant proteins of the present invention can be in pure or substantially pure form, and in the presence or absence of a stabilizing agent. Stabilizing agents include both proteinaceous or non-proteinaceous material and are well-known in the art. Stabilizing agents, such as albumin and polyethylene glycol (PEG) are known and are commercially available.

[00104] Although high levels of purity are preferred when the recombinant proteins of the present invention are used as therapeutic agents, the isolated proteins of the present invention are also useful in the presently disclosed methods at lower purity. For example, partially purified proteins of the present invention can be used as immunogens to raise antibodies in laboratory animals.

[00105] The recombinant renalase proteins of the present invention further may used in the preparation of pharmaceutical compositions, which may comprise, inter alia, a cofactor for enzyme activation. Renalase is a flavin-adenosine-dinucleotide (FAD)-containing enzyme that requires the cofactor FAD for its functionality. It will be readily apparent to one skilled in the art that other enzyme cofactors such as those which may function in a manner substantially similar to FAD and those well-known in the art can be employed to activate or deactivate renalase activity. These cofactors include, but are not limited to, FAD analogs, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), thiamine pyrophosphate, flavin mononucleotide (FMN), pyridoxal phosphate, coenzyme A, tetrahydro folate, adenosine triphosphate, guanosine triphosphate and S- adenosyl methionine (SAM), metal ion, metal porphyrin, e.g. heme groups, biotin, β2- microglobulin, thiamine pyrophosphate, coenzyme A, pyridoxal phosphate, coenzyme B 12, biocytine, tetrahydrofolate, and lipoic acid.

[00106] The recombinant renalase of the invention may exist as a multimer. Homodimers or homomultimers may be formed by a spontaneous association of two or several renalase subunits. There is evidence that renalase forms a dimer or multimer complex. It is believed that dimerization or multimerization of renalase may positively or negatively affect the enzyme activity. Accordingly, it is expected that disruption or stabilization of the dimer or multimer complex of renalase may be particularly useful for various therapeutic purposes.

[00107] In other related aspects, the invention includes methods of using an isolated nucleic acid encoding a mammalian renalase operably linked to a nucleic acid comprising a

54883 vl/DC 24 . -

promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses methods of using expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (1989, supra), and Ausubel et al. (1997, supra).

[00108] The vectors used in accordance with the present invention typically comprise an origin of replication, and the vector may or may not in addition comprise a "marker" or "selectable marker" function by which the vector can be identified and selected. While any selectable marker can be used, selectable markers for use in recombinant vectors are generally known in the art and the choice of the proper selectable marker will depend on the host cell. Examples of selectable marker genes which encode proteins that confer resistance to antibiotics or other toxins include, but are not limited to ampicillin, methotrexate, tetracycline, neomycin (Southern et al., J., J MoI Appl Genet. 1982; 1 (4):327-41 (1982)), mycophenolic acid (Mulligan et al., Science 209: 1422-7 (1980)), puromycin, zeomycin, hygromycin (Sugden et al., MoI Cell Biol. 5(2):410-3 (1985)) and G418. As will be understood by those of skill in the art, expression vectors typically include an origin of replication, a promoter operably linked to the coding sequence or sequences to be expressed, as well as ribosome binding sites, RNA splice sites, a polyadenylation site, and transcriptional terminator sequences, as appropriate to the coding sequence(s) being expressed.

[00109] Reference to a vector or other DNA sequences as "recombinant" merely acknowledges the operable linkage of DNA sequences that are not typically operably linked as isolated from or found in nature. Regulatory (expression and/or control) sequences are operatively linked to a nucleic acid coding sequence when the expression and/or control sequences regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression and/or control sequences can include promoters, enhancers, transcription terminators, a start codon (i.e., ATG) 5' to the coding sequence, splicing signals for introns and stop codons.

[00110] In one exemplary aspect of the invention, vector introduction or administration to a cell (transfection) is followed by one or more of the following steps:

54883 vi/DC 25 orney oc e o. -

[00111] (1 ) culturing the transfected cell under conditions suitable for selecting for a cell expressing the recombinant protein or polypeptide;

[00112] (2) evaluating expression of the recombinant protein or polypeptide; and

[00113] (3) collecting the recombinant protein or polypeptide.

[00114] In certain aspects of the present invention, the recombinant renalase produced is employed in a pharmaceutical composition. The composition may be used to treat, inter alia, end stage renal disease. Preferably, the composition comprises a pharmaceutically- acceptable carrier. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non toxic parenterally acceptable diluent or solvent, such as water or 1 ,3 butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di- glycerides.

[00115] Pharmaceutical compositions may be administered, prepared, packaged, and/or sold in formulations suitable for any method of administration, including oral, inhalation, or parenteral. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[00116] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[00117] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further

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depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[00118] As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

[00119] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen free water) prior to parenteral administration of the reconstituted composition.

[00120] As used herein, '"additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other

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"additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[00121] Typically, dosages of the compound of the invention which may be administered to an animal, preferably a human, will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.

[00122] The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[00123] The recombinant renalase proteins of the present invention may be used to treat, ameliorate, or prevent a number of conditions, diseases, or disorders.

[00124] In one aspect of the present invention, there is provided a method to treat diseases, disorders, and conditions associated with or mediated by mammalian renalase. Such disease, disorders and conditions include, but are not limited to, ESRD, chronic hypertension, systolic hypertension, isolated systolic hypertension, diabetic hypertension, pulmonary hypertension, acute severe hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure (CHF), myocardial infarction (MI), cardiac rhythm disturbance, stroke, atherosclerosis, depression, anxiety, mania and schizophrenia and the like.

[00125] Without further description, one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way.

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EXAMPLES

Method

[00126] Cloning of renalase gene into pPICZ (InVitrogen) vectors

[00127] The coding region of the human renalase gene was amplified by PCR and ligated into pPICZ vectors (INVITROGEN) in frame with the 3' c-myc epitope and polyhistidine tag.

[00128] For cloning into the pPICZα vector (INVITROGEN), the 5' PCR primer was designed to remove the first 3 amino acids of renalase (renalase construct pPICZα _ren_4- 342), and to clone renalase in-frame with the alpha factor, and immediately adjacent to the Kex2 signal cleavage. This construct takes advantage of the alpha factor signal sequence to facilitate renalase secretion into the medium upon cleavage of the alpha factor.

[00129] The renalase gene was also cloned into pPICZ, which lacks a signal sequence

(renalase construct pPICZ_ren_l-342). This construct was designed to test whether the native renalase signal peptide could mediate renalase secretion in a yeast expression system.

[00130] The constructs were amplified by transfection into E CoIi strains that are recombination deficient and deficient in endonuclease A (either DH5α or TOPOlO).

[00131] Transformation of P ichia Pastoris strains

[00132] Yeast strains X-33, GS l 15 and KM71H were transformed either by electroporation or using the EasyComp^{1 M} kit (InVitrogen) with either pPICZ_ren_ 1-342 or pPICZα _ren_4-342 that had been linearized at the Sad site.

[00133] Transformants were identified by their ability to grow in the presence of

Zeocin, and the correct integration of the renalase vector was confirmed by PCR.

[00134] Expression of recombinant renalase

[00135] Transformed clones were grown in 1 liter baffled flasks at 25-30⁰C, and the secretion of recombinant renalase into the medium was assessed by Western blotting starting on the 3^rd day post methanol induction, and for up to 8 days.

Results

[00136] Pichia strains X-33 and KM71H transformed with either pPICZ_ren_l -342 or pPICZα _ren_4-342 secreted renalase into the medium (Figures 1 and 2). Renalase secretion

54883 vl/DC 29 . -

progressively increased from day 3 to 8 and reached approximately 5 mg/L in the X-33 cultures. The secreted protein is stable and does not appear to undergo detectable proteolytic degradation. In vitro assays using the Amplex Red method indicate that the secreted protein is capable of metabolizing catecholamines.

Conclusions

[00137] We have described a method for synthesizing recombinant renalase using

Pichia Pastoris. The signal peptide of renalase is sufficient for secretion into the medium. Renalase secretion increases for at least 8 days with no evidence of protein degradation. A significant improvement in yield could be achieved by selecting clones with multiple integration events and by optimizing the fermentation conditions.

[00138] The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art.

[00139] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

54883 vl /DC 30

Claims

.CLAIMS

1. A method for producing recombinant renalase protein, the method comprising: culturing a Pichiapastoris harboring a recombinant renalase coding sequence operably linked to a promoter, and under conditions that permit expression of said renalase protein; and recovering the recombinant renalase protein.

2. The method of claim 1, wherein the renalase protein is a mammalian renalase protein.

3. The method of claim 1 , wherein the renalase protein is a human renalase protein.

4. The method of claim 1 , wherein expression of the renalase protein is induced by the presence of methanol in the culture medium.

5. The method of claim 1, wherein the renalase protein is active.

6. The method of claim 1 , wherein the renalase protein is recovered from the culture medium.

7. The method of claim 6, wherein the renalase protein is secreted into the culture medium via a P, pastoris alpha factor signal sequence.

8. The method of claim 7, wherein the renalase coding sequence is cloned in frame with the alpha factor signal sequence and adjacent to a Ke x2 cleavage signal.

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9. The method of claim 3, wherein the recombinant human renalase is recovered from the culture medium, and the recovered protein does not have the first 3 amino acids of human renalase.

10. The method of claim 6, wherein the renalase protein is secreted into the culture medium via a native renalase signal sequence.

1 1. The method of claim 4, wherein the promoter is an AOXl promoter.

12. The method of claim 1 , wherein the Pichia pastoris is cultured for up to eight days.

13. A recombinant renalase protein produced according to the method of claim 1.

14. A recombinant human renalase consisting of amino acids 4-342 of SEQ ID NO:2.

15. The recombinant renalase protein of claim 13 or 14, wherein the recombinant renalase protein is active,

16. A recombinant Pichia pastoris cell harboring a mammalian renalase coding sequence.

17. The recombinant Pichia pastoris cell of claim 16, wherein the cell secretes a renalase protein.

18. The recombinant cell of claim 16, wherein the cell secretes a renalase protein via the native renalase signal sequence.

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19. The recombinant cell of claim 16, wherein the cell secretes a renalase protein via cleavage of a P, pastoris alpha factor signal sequence.

20. The recombinant cell of claim 16, wherein the cell expresses renalase in the presence of methanol.

21. The recombinant cell of claim 20, wherein the cell contains a renalase coding sequence operably linked to an AOXl promoter

22. A recombinant vector comprising: a renalase coding sequence operably linked to a promoter, wherein the vector is suitable for expression of renalase in Pichia pastoris.

23. The recombinant vector of claim 22, wherein the renalase coding sequence is cloned in frame with a P. pastoris alpha factor signal sequence.

24. The recombinant vector of claim 22, wherein the renalase coding sequence encodes the native renalase signal sequence,

25. A method for producing recombinant renalase protein, the method comprising: culturing a methylotrophic yeast harboring a recombinant renalase coding sequence operably linked to a promoter, and under conditions that permit expression of said renalase protein; and recovering the recombinant renalase protein.

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