Ferroportin1 antibodies and methods Number:7,521,055 from the United States Patent and Trademark Office (PTO) owispatent

Title: Ferroportin1 antibodies and methods

Abstract: Positional cloning has been carried out to identify the gene responsible for the hypochromic anemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a novel multiple-transmembrane domain protein, expressed in the yolk sac. Zebrafish ferroportin1 is required for the transport of iron from maternally-derived yolk stores to the circulation, and functions as an iron exporter when expressed in Xenopus oocytes. Human and mouse homologs of the ferroportin1 gene have been identified. The invention includes isolated polynucleotides, vectors and host cells comprising nucleotide sequences encoding Ferroportin1 proteins and variants thereof, including those having iron transport function. The invention also includes polypeptides encoded by ferroportin1 genes and variants of such polypeptides, and fusion polypeptides comprising a Ferroportin1 or a portion thereof. Methods to produce a Ferroportin1, methods to produce antibodies to a Ferroportin1 and methods to identify agents binding to a Ferroportin1, which can be inhibitors or enhancers of Ferroportin1 iron transport activity, are also described. Inhibitors of Ferroportin1 activity can be used in a therapy for hemochromatosis.

Patent Number: 7,521,055 Issued on 04/21/2009 to Zon,   et al.

---

Inventors:	Zon; Leonard I. (Wellesley, MA), Donovan; Adriana (West Roxbury, MA)
Assignee:	Children's Medical Center Corporation (Boston, MA)
Appl. No.:	11/641,589
Filed:	December 19, 2006

---

Related U.S. Patent Documents

---

	Application Number	Filing Date	Patent Number	Issue Date
	09715927	Nov., 2000	7166448
	09567672	May., 2000
	60133382	May., 1999

---

Current U.S. Class:	424/184.1 ; 424/130.1; 424/178.1; 530/387.1; 530/387.3; 530/387.9; 530/388.1; 530/388.15; 530/388.22; 530/388.26; 530/389.1; 530/391.3
Current International Class:	A61K 39/00 (20060101); A61K 39/395 (20060101); C07K 16/00 (20060101); C12P 21/08 (20060101); C07K 14/00 (20060101); C07K 5/00 (20060101)

---

References Cited [Referenced By]

---

U.S. Patent Documents

6762293	July 2004	van Duijn et al.

Other References

Donovan, A., et al.; GenBank No. 7109244; Accession No. AF226612; Danio rerio ferroportin1 (fpn1) mRNA, complete coding sequence; Release Date: Feb. 29, 2000. cited by other . Donovan, A., et al.; GenBank No. 7109246; Accession No. AF22613; Mus musculus ferroportin1 (Fpn1)) mRNA, complete coding sequence; Release Date: Feb. 29, 2000. cited by other . Donovan, A., et al.; GenBank No. 7109248; Accession No. AF22614; Homo sapiens ferroportin1 (FPN1) mRNA, complete coding sequence; Release date: Feb. 29, 2000. cited by other . McKie, A. T., et al.; GenBank No. 7657099; Accession No. NM 014585; Homo sapiens ferroportin1; iron regulated gene 1 (FPN1), mRNA; Release date: Mar. 20, 2000. cited by other . Fujiwara, T., et al.; GenBank No. 966878; Accession No. D63209; HUM506F01B Clontech human placenta polyA+ mRNA (#6572) Homo sapiens cDNA clone GEN-506F01 5', mRNA sequence; Release Date: Aug. 29, 1995. cited by other . Marra, M., et al., GenBank No. 2235263; Accession No. AA500296; vi97h02.r1 Barstead mouse pooled organs MPLRB4 Mus musculus cDNA clone IMAGE:920211 5', mRNA sequence; Release Date: Jul. 1, 1997. cited by other . Hillier, L., et al.; GenBank No. 1300295; Accession No. W23461; zb33c01.r1 Soares parathyroid tumor NbHPA Homo sapiens cDNA clone IMAGE:305376 5', mRNA sequence; Release Date: May 6, 1996. cited by other . Hillier, L., et al.; GenBank No. 1278239; Accession No. W05488; za84h11.r1 Soares fetal lung NbHL19W Homo sapiens cDNA IMAGE:299301 5', mRNA sequence; Release Date: Apr. 23, 1996. cited by other . Kaplan, J., et al.; "Mining the genome for iron," Nature, 403:711-713 (2000). cited by other . Richardson, D.R., et al., "Another jigsaw piece towards solving the iron metabolism puzzle: the cloning of the iron exporter, ferroportin1," Redox Report, 5(1):7-9 (2000). cited by other . Griffiths, W., et al., "Haemochromatosis: novel gene discovery and the molecular pathophysiology of iron metabolism," Hum. Mol. Genet., 9(16):2377-2382 (2000). cited by other . Donovan, A., et al., "Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter," Nature, 403:776-781 (2000). cited by other . McKie, A., et al., "A Novel Duodenal Iron-Regulated Transporter, IREG1, Implicated in the Basolateral Transfer of Iron to the Circulation," Mol. Cell, 5: 299-309 (2000). cited by other . Abboud, S. and Haile, D. "A Novel Mammalian Iron-regulated Protein Involved in Intracellular Iron Metabolism," J. Biol. Chem. 275: 19906-19912 (2000). cited by other . Montosi, G., et al., "Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene," J. Clin. Invest. 108(4): 619-623 (2001). cited by other . Njajou, O.T., et al., "A mutation in SLC11A3 is associated with autosomal dominant hemochromatosis," Nature Genetics 28: 213-214 (2001). cited by other . GenBank accession No. NM.sub.--014585; Homo sapiens solute carrier family 40 (iron-regulated transporter), member 1 (SLC40A1), mRNA sequence; Release Date: Jun. 9, 2003. cited by other . Fleming, R. E. and Sly, W. S., "Ferroportin mutation in autosomal dominant hemochromatosis: loss of function, gain in understanding," J. Clin. Invest. 108(4): 521-522 (2001). cited by other . Bisson, L.F. and Coons, D.M., "Yeast Sugar Transporters," Crit. Rev. Biochem. Mol. Biol., 28(4):259-308 (1993). cited by other . Liang, H., et al., "Trinucleotide Insertions, Deletions, and Point Mutations in Glucose Transporters Confer K= Uptake in Saccharomyces cerevisiae," Mol. Cell. Biol. 18(2):926-935 (1998). cited by other . Dodsworth, S.J., et al., GenBank No. 1028149, Accession No. Z56918; H. sapiens CpG island DNA genomic Mse 1 fragment, clone 15368, forward read cpg 15368.ftla; Release Date: Oct. 19, 1995. cited by other . Arden, K.E., et al., "A Novel Mutation in Ferroportin 1 is Associated with Haemochromatosis in a Solomon Islands Patient," Gut, 52:1215-1217 (2003). cited by other.

Primary Examiner: Kemmerer; Elizabeth C.
Assistant Examiner: Wegert; Sandra
Attorney, Agent or Firm: Hamilton, Brook, Smith & Reynolds, P.C.

---

Government Interests

---

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant R01 DL53298-02 from the National Institutes for Health. The Government has certain rights in the invention.

---

Parent Case Text

---

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/715,927, now U.S. Pat. No. 7,166,448, filed Nov. 17, 2000, which is a continuation in-part of U.S. application Ser. No. 09/567,672, now abandoned, filed May 9, 2000 which claims the benefit of U.S. Provisional Application No. 60/133,382, filed on May 10, 1999. The entire teachings of the above applications are incorporated herein by reference.

---

Claims

---

What is claimed is:

1. A method for eliciting an immune response in an animal, said method comprising introducing into the animal a composition comprising a polypeptide comprising at least 19 consecutive amino acid residues of SEQ ID NO:2.

2. A method for eliciting an immune response in an animal, said method comprising introducing into the animal a composition comprising a polypeptide comprising at least 19 consecutive amino acid residues of SEQ ID NO:4.

3. A method for eliciting an immune response in an animal, said method comprising introducing into the animal a composition comprising a polypeptide comprising at least 19 consecutive amino acid residues of SEQ ID NO:6.

4. An antibody that binds specifically to at least one Ferroportin1 protein, wherein the amino acid sequence of the protein consists of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

5. A method for producing antibodies, said method comprising introducing into an animal isolated zebrafish Ferroportin1 protein, wherein the amino acid sequence of the protein consists of SEQ ID NO:2, or introducing into an animal an antigenic fragment of the zebrafish Ferroportin1 protein.

6. A method for producing antibodies, said method comprising introducing into a non-murine animal isolated mouse Ferroportin1 protein, wherein the amino acid sequence of the protein consists of SEQ ID NO:4, or introducing into a non-murine animal an antigenic fragment of the mouse Ferroportin1 protein.

7. A method for producing antibodies, said method comprising introducing into a non-human animal isolated human Ferroportin1 protein, wherein the amino acid sequence of the protein consists of SEQ ID NO:6, or introducing into a non-human animal an antigenic fragment of the human Ferroportin1 protein.

---

Description

---

BACKGROUND OF THE INVENTION

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMT1 (Fleming, M. D., et al., Nature Genet., 16:383-386, 1997; Gunshin, H., et al., Nature, 388:482-488, 1997; Andrews, N. C., N. Engl. J. Med., 341:1986-1995, 1999). A second transporter has been postulated to export iron across the basolateral surface to the circulation. The function of this iron transporting protein may be perturbed in mammalian disorders of iron deficiency or overload. Drugs to alter the function of this iron transporting protein may be useful to treat such diseases as hemochromatosis and some forms of anemia.

SUMMARY OF THE INVENTION

The invention relates to a number of nucleic acids, wherein the nucleic acids have SEQ ID NO:1, 3, 5 or 7 as described herein or the nucleic acids have nucleotide sequences related to those given specifically by SEQ ID NO by properties of hybridization, or by varying extents of identity, or by varying degrees of similarity as can be determined by a computer program designed for the purpose of comparing nucleotide or amino acid sequences. SEQ ID NO:1 is the nucleotide sequence of a cDNA encoding a zebrafish ferroportin1; SEQ ID NO:3 is the nucleotide sequence of a cDNA encoding a mouse ferroportin1; SEQ ID NO:5 is the nucleotide sequence of a cDNA encoding a human ferroportin1. SEQ ID NO:7 is the nucleotide sequence of a genomic DNA comprising the introns and exons of a human ferroportin1 gene. Also part of the invention are contiguous portions of any of the above nucleic acids, nucleic acids encoding any of the amino acid sequences described herein and nucleic acids encoding polypeptides which are variants of the Ferroportin1 proteins described herein by amino acid sequence. Further nucleic acids which are part of the invention are those encoding a fusion polypeptide comprising a Ferroportin1 or a portion of a Ferroportin1.

Related to the isolated nucleic acids are vectors and host cells comprising nucleotide sequences identical to the isolated nucleic acids of the invention. In some cases, regulatory sequences can be operably linked to coding regions to allow expression of a gene. Such cells can be maintained under conditions in which the gene is expressed and the encoded polypeptide is produced. The polypeptide can be purified by one or more steps to increase the proportion of polypeptide in the milieu of medium and material of cellular origin, thereby producing isolated polypeptide.

The term regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals and enhancers, for instance.

The invention also includes Ferroportin1 proteins, for example, those having amino acid sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, proteins which are naturally occurring mutants or variants of those proteins characterized by those specific amino acid sequences, and mutants and variants of those proteins identified as having the specific amino acid sequence SEQ ID NO:2, 4, or 6 that are produced by laboratory manipulations of the nucleic acids encoding a Ferroportin1. Also within the invention are contiguous portions of any of the polypeptides with SEQ ID NO:2, 4, or 6, or portions of such mutants or variants of the polypeptides described herein as containing amino acid substitutions, or described herein as having a certain percent identity or similarity to another sequence in a comparison. A further embodiment of the invention is a fusion polypeptide, which can comprise a Ferroportin1 of full-length amino acid sequence, as in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or a contiguous portion thereof, or can comprise any of the mutants or variant polypeptides, or portions thereof, as described herein, for example by their amino acid sequence identity or similarity to an amino acid sequence identified by SEQ ID NO, or by their activity (e.g., iron transport function) or property of binding to antibodies produced by immunizing an animal with a Ferroportin1.

Antibodies that bind to one or more Ferroportin1 proteins are also an aspect of the invention. Antibodies to a Ferroportin1 of one or more species can be produced, for example, by introducing into an animal which is not the source of the Ferroportin1 immunogen a Ferroportin1 or an immunogenic portion thereof in a suitable medium, which can include such substances as stabilizing agents and adjuvant. Other known methods can be used to make hybridomas producing monoclonal antibodies that bind to one or more Ferroportin1 proteins, as isolated, or as they exist in a cell membrane.

Other aspects of the invention include methods for identifying agents which bind to a Ferroportin1 (or to a mutant, variant, Ferroportin1 fusion protein or a contiguous portion of any of the foregoing) by steps that include contacting the agent with the isolated protein under conditions appropriate for binding of the agent to the isolated protein, and detecting a resulting agent-protein complex. Similar methods can be used to identify an agent which is an inhibitor or an enhancer of a function of a Ferroportin1 protein, where the steps can be the following: (a) combining (1) said isolated protein; (2) the ligand of said protein; and (3) a candidate agent to be assessed for its ability to inhibit interaction between said protein of (1) and the ligand of (2), under conditions appropriate for interaction between the said protein of (1) and the ligand of (2); (b) determining the extent to which said protein of (1) and the ligand of (2) interact; and (c) comparing the extent determined in (b) with the extent to which interaction of said protein of (1) and the ligand of (2) occurs in the absence of the candidate agent to be assessed and under the same conditions appropriate for interaction of said protein of (1) with the ligand of (2); wherein if the extent to which interaction of said protein of (1) and the ligand of (2) occurs is less in the presence of the candidate agent than in the absence of the candidate agent, the candidate agent is an agent which inhibits interaction between said protein and the ligand of said protein. If greater export of the iron from the test cells compared to export of the iron from the control cells is observed, this is indicative that the agent is an enhancer of iron export by said protein. An agent can be further tested for its effect on a Ferroportin1 protein in an animal, if the following steps are carried out: a) administering the agent to one or more test animals; b) measuring exogenously supplied iron in one or more samples of tissue or bodily fluid from said test animals; c) measuring exogenously supplied iron in one or more comparable samples of tissue or bodily fluid from suitable control animals; and d) comparing the iron of b) with the iron of c); whereby, lower iron in step b) than in step c) is indicative that the agent is an inhibitor of said protein. An inhibitor of the iron transport function of a human Ferroportin1 can be used in a method for treating hemochromatosis in a human, said method comprising administering to the human an inhibitor of Ferroportin1 iron transport function, or such inhibitor can be used in a method for treating a disease or medical disorder resulting from oxidative damage in a mammal, said method comprising administering to the mammal an inhibitor of Ferroportin1 iron transport function. Enhancers of a Ferroportin1 can be used in a method for treating iron deficiency anemia in a mammal, said method comprising administering to the mammal an enhancer of Ferroportin1 iron transport function.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

FIG. 1 is a map of the weh locus showing positional cloning of the weissherbst gene. The weh locus is depicted by a thick black bar just distal to the AFLP marker I36. Below the map is an enlarged view of the weh locus that depicts the BAC and PAC genomic clones identified by a chomosomal walk in an analysis of 3873 meioses. Genotyping of a total of 1783 meioses from haploid animals and 2090 meioses from diploid mutants narrowed the critical interval containing the gene to the PAC clones 211O13 and 170G3. The numbers of recombination events identified on the proximal side (circles) and distal side (squares) of the weh locus are indicated.

FIG. 2 is an amino acid sequence alignment of zebrafish (SEQ ID NO: 2), human (SEQ ID NO: 6) and mouse (SEQ ID NO: 4) Ferroportini (FPN1). The initiator methionine in all three species was established by the presence of upstream, in-frame stop codons. Shading indicates identical amino acids. Bars under sequence indicate predicted transmembrane domains. The mutations identified in the weh.sup..about.p85c and weh.sup.th2.about.8 alleles are indicated by black circles below the affected amino acids.

FIG. 3 is a bar graph showing the measurement of iron efflux from Xenopus oocytes. Oocytes expressing either DMT1 alone or DMT1 and FPN1 were loaded with .sup.55Fe by incubation in uptake buffer containing 60 .mu.M .sup.55FeCl.sub.2. Efflux from individual oocytes was measured by incubation of oocytes in 500 .mu.l of efflux buffer with or without 20 mg/ml apo-transferrin (-apoTfr and +apoTfr). After efflux, the total .sup.55Fe content of both the efflux solution and the individual oocytes was measured by scintillation counting. The data are expressed as an average (n=6) of the ratio of the pmols of efflux to the pmols of uptake per oocyte.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Iron is required for many cellular processes, but it can also be toxic when present in excess. Thus, iron homeostasis must be strictly maintained. Studies described herein employed zebrafish genetics to identify the multiple-transmembrane domain protein Ferroportin1, an iron export protein. In the mammalian yolk sac and placenta, Ferroportin1 may play an important conserved role in the transport of iron from the maternal to the embryonic circulation. In adults, Ferroportin1 is likely to function in iron transport at the basolateral surface of duodenal enterocytes. In disorders such as iron deficiency or overload, tissues respond by altering normal iron utilization. Ferroportin1 could be involved in the pathophysiology of iron deficiency anemias or iron overload syndromes, such as hemochromatosis.

As described herein, Ferroportin1 refers to an evolutionarily conserved family of proteins that mediate the transport of iron out of cells. The family includes proteins which are conserved at least as widely as from zebrafish to humans and exhibit very different expression patterns in tissues. Specific embodiments described include Ferroportin1 proteins from mice, humans and zebrafish which have been shown to be functional iron transporters. The term Ferroportin1 can refer to other proteins sharing at least about 70% sequence similarity, more preferably at least about 80% sequence similarity, and still more preferably, at least about 90% sequence similarity, and most preferably, at least about 95% sequence similarity.

One aspect of the invention relates to isolated nucleic acids or polynucleotides that encode a Ferroportin1 as described herein, such as those Ferroportin1 proteins having an amino acid sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 and nucleic acids closely related thereto as described herein.

Using the information provided herein, such as a nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, a nucleic acid of the invention encoding a Ferroportin1 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing cDNA library fragments, followed by obtaining a full length clone. For example, to obtain a nucleic acid of the invention, a library of clones of cDNA of a species of animal can be probed with a labeled oligonucleotide, such as a radiolabeled oligonucleotide, preferably about 17 nucleotides or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent (also, "high stringency") hybridization conditions. By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full length sequence. Suitable techniques are described, for example, in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds.), containing supplements through Supplement 49, 2000, John Wiley and Sons, Inc., especially chapters 5, 6 and 7.

Embodiments of the invention include isolated nucleic acid molecules comprising any of the following nucleotide sequences: 1.) a nucleotide sequence which encodes a protein comprising the amino acid sequence of human Ferroportin1 (SEQ ID NO:6), the amino acid sequence of mouse Ferroportin1 (SEQ ID NO:4), or the amino acid sequence of zebrafish Ferroportin1 (SEQ ID NO:2); 2.) nucleotide sequences of human ferroportin1, mouse ferroportin1, or zebrafish ferroportin1; 3.) a nucleotide sequence which is complementary to the nucleotide sequence of human ferroportin1 (SEQ ID NO:5), mouse ferroportin1 (SEQ ID NO:3), zebrafish ferroportin1 (SEQ ID NO:1); 4.) a nucleotide sequence which consists of the coding region of human ferroportin1 (within SEQ ID NO:5), the coding region of mouse ferroportin1 (within SEQ ID NO:3), or the coding region of zebrafish ferroportin1 (within SEQ ID NO:1).

The invention further relates to nucleic acids (nucleic acid molecules or polynucleotides) having nucleotide sequences identical over their entire length to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO: 5. It further relates to DNA, which due to the degeneracy of the genetic code, encodes a Ferroportin1 protein whose amino acid sequence is provided herein. Also provided by the invention are nucleic acids having the coding sequences for the mature polypeptides or fragments in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence. The nucleic acids of the invention encompass nucleic acids that include a single continuous region or discontinuous regions encoding the polypeptide, together with additional regions, that may also contain coding or non-coding sequences. The nucleic acids may also contain non-coding sequences, including, for example, but not limited to, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequences which encode additional amino acids.

The nucleic acid molecules of the invention can comprise, in addition to sequences identified by SEQ ID NO or sequences related to these by variations and by hybridization as described herein, other sequences encoding unrelated (heterologous--that is, with insignificant sequence similarity to a Ferroportin1) polypeptides or peptides. These peptides or polypeptides can be whole proteins, as occur naturally or as have been modified by design. Together, the nucleic acid sequences make up genes for hybrid or fusion proteins. For example, an unrelated marker sequence that facilitates purification (e.g., by affinity column) of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence can be a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767 (1984)), or a sequence encoding glutathione S-transferase of Schistosoma japonicum (vectors available from Pharmacia; see Smith, D. B. and Johnson K. S., Gene 67:31 (1988) and Kaelin, W. G. et al., Cell 70:351 (1992)). For additional applications, the unrelated nucleic acid sequence can encode a peptide or polypeptide which is immunogenic or which enhances the immunogenicity of the fusion protein or polypeptide. Nucleic acids of the invention also include, but are not limited to, nucleic acids comprising a structural gene and its naturally associated sequences that control gene expression.

The invention further relates to variants, including naturally-occurring allelic variants, of those nucleic acids described specifically herein by DNA sequence, that encode variants of such polypeptides as those having the amino acid sequences SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Such variants include nucleic acids encoding variants of the above-listed amino acid sequences, wherein those variants have several, such as 5 to 10, 1 to 5, or 3, 2 or 1 amino acids substituted, deleted, or added, in any combination. Variants include polynucleotides encoding polypeptides with at least 95% but less than 100% amino acid sequence identity to the polypeptides described herein by amino acid sequence. Variant polynucleotides hybridize, under low to high stringency conditions, to the alleles described specifically herein by DNA sequence. In one embodiment, variants have silent substitutions, additions and deletions that do not alter the properties and activities of the Ferroportin1. Allelic variants of the polynucleotides encoding human Ferroportin1 (SEQ ID NO:5), mouse Ferroportin1 (SEQ ID NO:3), and zebrafish Ferroportin1 (SEQ ID NO:1) will be identified as mapping to chromosomal locations corresponding to the chromosomal locations of the wild type genes.

Orthologous genes are gene loci in different species that are sufficiently similar to each other in their nucleotide sequences to suggest that they originated from a common ancestral gene. Orthologous genes arise when a lineage splits into two species, rather than when a gene is duplicated within a genome. Proteins that are orthologs are encoded by genes of two different species, wherein the genes are said to be orthologous.

The invention further relates to polynucleotides encoding polypeptides which are orthologous to those polypeptides having a specific amino acid sequence described herein, such as the amino acid sequences (SEQ ID NO:2), (SEQ ID NO:4), and (SEQ ID NO:6). These polynucleotides, which can be called ortholog polynucleotides, encode orthologous polypeptides that can range in amino acid sequence identity to a reference amino acid sequence described herein, from about 65% to less than 100%, but preferably 70% to 80%, more preferably 80% to 90%, and still more preferably 90% to less than 100%. Orthologous polypeptides can also be those polypeptides that range in amino acid sequence similarity to a reference amino acid sequence described herein from about 75% to 100%. The ortholog polynucleotides encode polypeptides that have similar functional characteristics (e.g., iron transport activity) and similar tissue distribution, as appropriate to the organism from which the ortholog polynucleotides can be isolated.

Ortholog polynucleotides can be isolated from (e.g., by cloning or nucleic acid amplification methods) a great number of species, as shown by the sample of Ferroportin1 proteins from evolutionarily divergent species described herein. Ortholog polynucleotides corresponding to SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 are those which can be isolated from mammals such as rat, dog, chimpanzee, monkey, baboon, pig, rabbit and guinea pig, for example.

Further variants that are fragments of the nucleic acids of the invention may be used to synthesize full-length nucleic acids of the invention, such as by use as primers in a polymerase chain reaction. As used herein, the term primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes. The term primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.

Further embodiments of the invention are nucleic acids that are at least 80% identical over their entire length to a nucleic acid described herein, for example a nucleic acid having the nucleotide sequence in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. Additional embodiments are nucleic acids, and the complements of such nucleic acids, having at least 90% nucleotide sequence identity to the above-described sequences, and nucleic acids having at least 95% nucleotide sequence identity. In preferred embodiments, DNA of the present invention has 97% nucleotide sequence identity, 98% nucleotide sequence identity, or at least 99% nucleotide sequence identity with the DNA whose sequences are presented herein.

Other embodiments of the invention are nucleic acids that are at least 80% identical in nucleotide sequence to a nucleic acid encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, and nucleic acids that are complementary to such nucleic acids. Specific embodiments are nucleic acids having at least 90% nucleotide sequence identity to a nucleic acid encoding a polypeptide having an amino acid sequence as described in the list above, nucleic acids having at least 95% sequence identity, and nucleic acids having at least 97% sequence identity. Also included in the invention are nucleic acid molecules comprising at least 80%, 90%, 95%, or 97% of the coding region of any of SEQ ID NOs 1, 3 or 5.

The terms "complementary" or "complementarity" as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be "partial" in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single-stranded molecules (that is, when A-T and G-C base pairing is 100% complete). The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend on binding between nucleic acid strands.

The invention further includes nucleic acids that hybridize to the above-described nucleic acids, especially those nucleic acids that hybridize under stringent hybridization conditions. Preferred nucleic acid molecules meeting these hybridization criteria also encode a polypeptide having an iron transport function. "Stringent hybridization conditions" or "high stringency conditions" generally occur within a range from about T.sub.m minus 5.degree. C. (5.degree. C. below the strand dissociation temperature or melting temperature (T.sub.m) of the probe nucleic acid molecule) to about 20.degree. C. to 25.degree. C. below T.sub.m. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect molecules having identical or related polynucleotide sequences. An example of high stringency hybridization follows. Hybridization solution is (6.times.SSC/10 mM EDTA/0.5% SDS/5.times. Denhardt's solution/100 .mu.g/ml sheared and denatured salmon sperm DNA). Hybridization is at 64-65.degree. C. for 16 hours. The hybridized blot is washed two times with 2.times.SSC/0.5% SDS solution at room temperature for 15 minutes each, and two times with 0.2.times.SSC/0.5% SDS at 65.degree. C., for one hour each. Further examples of high stringency conditions can be found on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., containing supplements up through Supplement 49, 2000). Examples of high, medium, and low stringency conditions can be found on pages 36 and 37 of WO 98/40404, which are incorporated herein by reference.

The invention further relates to nucleic acids obtainable by screening an appropriate library with a probe having a nucleotide sequence such as that set forth in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, or a probe which consists of the coding region of any of these SEQ ID NOs, or a probe which is a sufficiently long fragment of any of the above; and isolating the nucleic acid. Such probes generally can comprise at least 15 nucleotides. Nucleic acids obtainable by such screenings may include RNAs, cDNAs and genomic DNA, for example, encoding iron transport proteins of the Ferroportin1 protein family described herein.

Other nucleic acid embodiments are those comprising a nucleotide sequence encoding a contiguous portion of a polypeptide represented as having amino acid sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, wherein the portion is at least about 15 amino acids long, but can alternatively be at least 30 amino acids long or 60 amino acids long. The portion can be derived from amino acid sequence at the N-terminal, C-terminal or internal regions of SEQ ID NOs 2, 4 or 6.

Further uses for the nucleic acid molecules of the invention, whether encoding a full-length Ferroportin1 protein or whether comprising a contiguous portion of a nucleic acid molecule such as one given in SEQ ID NO:1, 3 or 5, include use as markers for tissues in which the encoded protein is preferentially expressed (to identify constitutively expressed proteins or proteins produced at a particular stage of tissue differentiation or stage of development of a disease state); as molecular weight markers on southern gels; as chromosome markers or tags (when labeled, for example with biotin, a radioactive label or a fluorescent label) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in a mammal to identify potential genetic disorders; as probes to hybridize and thus identify, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel nucleic acid molecules; for selecting and making oligomers for attachment to a "gene chip" or other support, to be used, for example, for examination of expression patterns in embryonic development or in organs of an animal at a particular developmental stage.

Further methods to obtain nucleic acids encoding Ferroportin1 proteins include PCR and variations thereof (e.g., "RACE" PCR and semi-specific PCR methods). Portions of the nucleic acids having a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, (especially "flanking sequences" on either side of a coding region) can be used as primers in methods using the polymerase chain reaction, to produce DNA from an appropriate template nucleic acid.

Once a fragment of the ferroportin1 gene is generated by PCR, it can be sequenced, and the sequence of the product can be compared to other DNA sequences, for example, by using the BLAST Network Service at the National Center for Biotechnology Information. The boundaries of the open reading frame can then be identified using semi-specific PCR or other suitable methods such as library screening. Once the 5' initiator methionine codon and the 3' stop codon have been identified, a PCR product encoding the full-length gene can be generated using cDNA as a template (the cDNA being generated from mRNA), with primers complementary to the extreme 5' and 3' ends of the gene or to their flanking sequences. The full-length genes can then be cloned into expression vectors for the production of functional proteins.

In some embodiments of the invention, the nucleic acid molecules can be modified at the base moiety, sugar moiety or phosphate backbone to change the stability, hybridization or solubility properties of the molecules. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids, or PNAs (Hyrup et al., Bioorganic and Medicinal Chemistry 4 :5-23, 1996). PNAs are nucleic acid mimics in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described by Hyrup et al. (1996) and Perry-O'Keefe et al., Proc. Natl. Acad. Sci. USA 93:14670-14675, 1996. PNAs can be used in place of nucleic acids for some applications, for example, as probes or primers for DNA sequence analysis and hybridization, or as antisense agents for sequence-specific modulation of gene expression.

Nucleic acid molecules of the present invention can be incorporated into various constructs (e.g., plasmids, bacteriophages, viruses, artificial chromosomes) and incorporated into host cells in these constructs or in one or more chromosomes of the host cell, for example, for further manipulation of the sequences or for production of an encoded polypeptide under suitable conditions for the growth or maintenance of the cells.

A host cell is a cell, or a descendant thereof, which has been transfected by an exogenous DNA sequence using methods within the skill of those in the art. See, e.g., Graham et al. (1973) Virology 52:456, Sambrook et al. (1989) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. More particularly, there are two major steps in transfection: first, the exogenous DNA must traverse the recipient (host) cell plasma membrane in order to be exposed to the cell's transcription and replication machinery; and second, the DNA must either become stably integrated into the host cell genome, or be capable of extra-chromosomal replication at a sufficient rate. A number of transfection methods have been described in the art, such as calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73).

The invention also relates to isolated proteins or polypeptides such as those encoded by nucleic acids of the present invention. Isolated proteins can be purified from a natural source or can be made recombinantly. Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides that exist in a state different from the state in which they exist in cells in which they are normally expressed in an organism, and include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, and also include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Thus, the term "isolated" as used herein, indicates that the polypeptide in question exists in a physical milieu distinct from that in which it occurs in nature. Thus, "isolated" includes existing in membrane fragments and vesicles, membrane fractions, liposomes, lipid bilayers and other artificial membrane systems. An isolated Ferroportin1 may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, and may even be purified essentially to homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC), but may also have further cofactors or molecular stabilizers, such as detergents, added to the purified protein to enhance activity. In one embodiment, proteins or polypeptides are isolated to a state at least about 75% pure; more preferably at least about 85% pure, and still more preferably at least about 95% pure, as determined by Coomassie blue staining of proteins on SDS-polyacrylamide gels. Proteins or polypeptides referred to herein as "recombinant" are proteins or polypeptides produced by the expression of recombinant nucleic acids.

"Polypeptide" as used herein indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term is also intended to include polypeptide that have been subjected to post-expression modifications such as, for example, glycosylations, acetylations, phosphorylations and the like.

In a preferred embodiment, an isolated polypeptide comprising a Ferroportin1, a functional portion thereof, or a functional equivalent of the Ferroportin1, has at least one function characteristic of a Ferroportin1, for example, transport activity, binding function (e.g., a domain which binds to a cofactor), or antigenic function (e.g., binding of antibodies that also bind to a naturally-occurring Ferroportin1, as that function is found in an antigenic determinant). Functional equivalents can have activities that are quantitatively similar to, greater than, or less than, the reference protein. These proteins include, for example, naturally occurring Ferroportin1 proteins that can be purified from tissues in which they are produced (including polymorphic or allelic variants), variants (e.g., mutants) of those proteins and/or portions thereof. Such variants include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides in which one or more residues are modified, and mutants comprising one or more modified residues. The portions of the invention also include isolated polypeptides encoded by a nucleic acid molecule, wherein said nucleic acid molecule hybridizes to a complement of any of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 under high stringency conditions. Portions or fragments of a Ferroportin1 can range in size from ten amino acid residues to the entire amino acid sequence minus one amino acid. An isolated polypeptide comprising a functional portion of a Ferroportin1 can comprise at least 10 amino acid residues of a cytoplasmic or extracellular domain of a Ferroportin1.

The isolated proteins of the invention preferably include mammalian iron transport proteins of the Ferroportin1 family of homologous proteins. In preferred embodiments, the extent of amino acid sequence identity between a polypeptide having one of the amino acid sequences SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, and the respective functional equivalents of these polypeptides is at least about 80% or 88%. In other embodiments, the degree of amino acid sequence identity between a Ferroportin1 and its respective functional equivalent is at least about 91%, at least about 94%, or at least about 97%.

The polypeptides of the invention also include those Ferroportin1 proteins encoded by polynucleotides which are orthologous to those polynucleotides, the sequences of which are described herein in whole or in part. Ferroportin1 proteins which are orthologs to those described herein by amino acid sequence, in whole or in part, are, for example, Ferroportin1 proteins of dog, rat, chimpanzee, monkey, rabbit, guinea pig, baboon and pig, and are also embodiments of the invention.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison. In the simplest concept of identity, two nucleic acid sequences or two amino acid sequences are compared after aligning them for the maximum number of matches at the same position, without the introduction of any gaps. In a somewhat more complex concept of identity, the sequences are aligned and gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment, and non-homologous (dissimilar) sequences can be disregarded for comparison purposes. In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity in terms of structure and chemical characteristics so as to perform one or more of the same functions performed by the polypeptides described herein by amino acid sequence. Similarity for a polypeptide is determined by amino acid substitutions, which can be conservative amino acid substitutions. For example, the invention encompasses polypeptides with at least one conservative amino acid substitution. Conservative substitutions are those that replace a given amino acid residue in a polypeptide with another amino acid residue of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr and Trp. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990).

TABLE-US-00001 TABLE Conservative Amino Acid Substitutions Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

The comparison of sequences and determination of percent similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereaux, J., eds., M. Stockton Press, New York, 1991). In a preferred embodiment, the percent similarity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison Wis., using, for example, a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent similarity between two nucleotide sequences is determined using the GAP program in the Wisconsin Package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent similarity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acids and protein sequences of the present invention can further be used as a "query sequence" to perform a search against databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTN, BLASTP, BLASTX, TBLASTN, TBLASTX programs (version 2.0) or PSI-BLAST 2.1 programs based on Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the BLASTN program, for example, with default parameters matrix=BIOSUM62, gap existence cost=11, per residue gap cost=1, lambda ratio=0.85, filtered, to obtain nucleotide sequences homologous to (with calculatably significant similarity to) the nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTP program, for example, with default parameters scoring matrix=BIOSUM62, word size=3, E value=10, gap costs=11,1 and alignments=50, to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Within the invention are isolated nucleic acid molecules having at least 80%, 85%, 90%, 95% and 97% sequence similarity to a nucleic acid encoding a polypeptide comprising the amino acid sequence SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. Also within the invention are isolated nucleic acid molecules which hybridize under high stringency conditions to nucleic acid consisting of the coding regions of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

The invention further relates to fusion proteins, comprising a Ferroportin1 or functional portion thereof (as described above) as a first moiety, linked to second moiety or to multiple moieties not occurring in the Ferroportin1 as found in nature. Thus, a second moiety can be an amino acid, peptide or polypeptide. The second moiety can be in an N-terminal location, C-terminal location or internal to the fusion protein, or multiple heterologous moieties can be in multiple locations. In one embodiment, the fusion protein comprises a Ferroportin1 or portion thereof having iron transport function as the first moiety, and a second moiety comprising a linker sequence and an affinity ligand. Fusion proteins can be produced by a variety of methods. For example, a fusion protein can be produced by the insertion of a ferroportin1 gene or portion thereof into a suitable expression vector, such as Bluescript SK+/-(Stratagene), pGEX-4T-2 (Pharmacia), pET-24(+) (Novagen), or vectors of similar construction. The resulting construct can be introduced into a suitable host cell for expression. Upon expression, fusion protein can be purified from cells by means of a suitable affinity matrix (See e.g., Current Protocols in Molecular Biology, Ausubel, F. M. et al., eds., Vol. 2, pp. 16.4.1-16.7.8, containing supplements up through Supplement 49, 2000).

The invention also relates to enzymatically produced, synthetically produced, or recombinantly produced portions of a Ferroportin1 protein. Portions of a Ferroportin1 can be made which have full or partial function on their own, or which when mixed together (though fully, partially, or nonfunctional alone), spontaneously assemble with one or more other polypeptides to reconstitute a functional protein having at least one function characteristic of a Ferroportin1.

Fragments of a Ferroportin1 can be produced by direct peptide synthesis, for example those using solid-phase techniques (Roberge, J. Y. et al., Science 269:202-204 (1995); Merrifield, J., J. Am. Chem. Soc. 85:2149-2154 (1963)). Peptide or polypeptide synthesis can be performed using manual techniques or by automation. Automated synthesis can be carried out using, for instance, an Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments of a Ferroportin1 can be synthesized separately and combined using chemical methods.

One aspect of the invention is a peptide or polypeptide having the amino acid sequence of a portion of a Ferroportin1 protein which is hydrophilic rather than hydrophobic, and ordinarily can be detected as facing the outside of the cell membrane. Such a peptide or polypeptide can be thought of as being an extracellular domain of the Ferroportin1, or a mimetic of said extracellular domain. Peptides or polypeptides comprising at least 10 amino acid residues of a cytoplasmic or extracellular domain of human, mouse or zebrafish Ferroportin1 can be synthesized.

The term "mimetic" as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of the Ferroportin1 of interest, or one or more portions thereof, and, as such, is able to effect some or all of the functions of a Ferroportin1.

Portions of a Ferroportin1 can be prepared by enzymatic cleavage of the isolated protein, or can be made by chemical synthesis methods. Portions of a Ferroportin1 can also be made by recombinant DNA methods in which restriction fragments, or fragments that may have undergone further enzymatic processing, or synthetically made DNAs are joined together to construct an altered ferroportin1 gene. The gene can be made such that it encodes one or more desired portions of a Ferroportin1. These portions of Ferroportin1 can be entirely homologous to a known Ferroportin1, or can be altered in amino acid sequence relative to naturally occurring Ferroportin1 proteins to enhance or introduce desired properties such as solubility, stability, or affinity to a ligand. A further feature of the gene can be a sequence encoding an N-terminal signal peptide directed to the plasma membrane.

Another aspect of the invention relates to a method of producing a Ferroportin1 protein, variants or portions thereof, and to expression systems and host cells containing a vector appropriate for expression of a Ferroportin1 protein.

Cells that express a Ferroportin1, a variant or a portion thereof, or an ortholog of a Ferroportin1 described herein by amino acid sequence, can be made and maintained in culture, under conditions suitable for expression, to produce protein in the cells for cell-based assays, or to produce protein for isolation. These cells can be procaryotic or eucaryotic. Examples of procaryotic cells that can be used for expression include Escherichia coli, Salmonella typhimurium and Bacillus subtilis. Examples of eucaryotic cells that can be used for expression include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lower eucaryotic cells, and cells of higher eucaryotes such as those from insects and mammals, such as primary cells and cell lines such as CHO, HeLa, 3T3, BHK, COS, human kidney 293 and Jurkat cells. (See, e.g., Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, Inc., containing Supplements up through Supplement 49, 2000)).

In one embodiment, host cells that produce a recombinant Ferroportin1, or a portion thereof, a variant, or an ortholog of a Ferroportin1 described herein by amino acid sequence, can be made as follows. A gene encoding a Ferroportin1, variant or a portion thereof can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, phage, cosmid, phagemid, virus, virus-derived vector (e.g., SV40, vaccinia, adenovirus, fowl pox virus, pseudorabies viruses, retroviruses) or other suitable replicon, which can be present in a single copy or multiple copies, or the gene can be integrated in a host cell chromosome. A suitable replicon or integrated gene can contain all or part of the coding sequence for a Ferroportin1 or variant, operably linked to one or more expression control regions whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation. The vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transfection, electroporation, infection). For expression from the Ferroportin1 gene, the host cells can be maintained under appropriate conditions (e.g., in the presence of inducer, normal growth conditions, etc.). Proteins or polypeptides thus produced can be recovered (e.g., from the cells, as in a membrane fraction, from the periplasmic space of bacteria, from culture medium) using suitable techniques. Appropriate membrane targeting signal peptides may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signal peptides that do no naturally occur with a Ferroportin1.

Polypeptides of the invention can be recovered and purified from cell cultures (or from their primary cell source) by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and high performance liquid chromatography. Known methods for refolding protein can be used to regenerate active conformation if the polypeptide is denatured during isolation or purification.

The host cells of the invention can be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which Ferroportin1 coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous ferroportin1 sequences have been introduced into their genome, or homologous recombinant animals in which endogenous ferroportin1 sequences have been altered. Such animals are useful for studying the function and/or activity of Ferroportin1, and for identifying and/or evaluating modulators of Ferroportin1 activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. "Exogenous" as used in the context of a transgenic animal, means different from that of the unaltered recipient host cell. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous weh gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing Ferroportin1 encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The ferroportin1 cDNA sequence can be introduced as a transgene into the genome