EP0971957A1 - Polypeptide exer ant une activite de regulation de l'appetit - Google Patents

Polypeptide exer ant une activite de regulation de l'appetit

Info

Publication number
EP0971957A1
EP0971957A1 EP98909375A EP98909375A EP0971957A1 EP 0971957 A1 EP0971957 A1 EP 0971957A1 EP 98909375 A EP98909375 A EP 98909375A EP 98909375 A EP98909375 A EP 98909375A EP 0971957 A1 EP0971957 A1 EP 0971957A1
Authority
EP
European Patent Office
Prior art keywords
leu
cys
lys
gly
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98909375A
Other languages
German (de)
English (en)
Inventor
Sven Hastrup
Kennet Christiansen
Lars Thim
Martin Edward Judge
Peter Kristensen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0971957A1 publication Critical patent/EP0971957A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a polypeptide with appetite regulating function/activity, a nucleic acid construct encoding the polypeptide and a method of producing the polypeptide.
  • the invention further relates to recombinant vectors comprising the nucleic acid construct encoding the polypeptide, recombinant host cells comprising the nucleic acid construct or the recombinant vector, a trans genic animal or plant containing and expressing the nucleic acid construct, an appetite regulating composition comprising the polypeptide, and the use of the polypeptide for regulating appetite.
  • the polypeptide has appetite regulating activity /function in mammals, including humans.
  • the aim of this invention has been to find the factor(s) responsible for this characteristic phenotype.
  • CART Cocaine and Amphetamine Regulated Transcript
  • a polypeptide of at least 30 amino acids was found by Spiess et al., 1981, Biochemistry 20: 1982-1988 as an HPLC peak when purifying somatostatine from sheep hypothalamus.
  • the isolated polypeptide was the C-terminal (IPI-CART) portion of CART. However, no biological function was associated with this molecule.
  • the mature CART peptide has so far not been isolated and characterised.
  • a transcript to be upregulated in rat brain after treatment with cocaine and amphetamine relating to CART was cloned. This cloning indicates that the peptide may exist in a long form consisting of 102 amino acid residues or in a short form consisting of 89 amino acid residues (Douglass, J. et al. J.Neuwsci. 15, 2471 -2481, 1995).
  • the same group found the human gene and cDNA for CART. Only the short form exists in humans (Douglass and Daoud (1996), Gene 169: 241-245).
  • the aim of this invention has been to find the factor(s) responsible for the above described characteristic phenotype.
  • the peptides SEQ ID Nos. 5 to 11 are considered to be novel per se and are constituting a part of the invention.
  • cysteine residues of the above peptides SEQ ID Nos. 1 to 11 are linked by disulphide bonds in the configuration I-III, II-V and IV-VI when the cysteines are numbered from the N-terminal.
  • These peptides are also considered to be novel per se and are constituting a part of the invention.
  • appetite regulating activity /function is intended to mean any activity/function which suppresses appetite e.g. by inducing a feeling of satiety or by inhibiting the sensation of hunger.
  • the appetite regulating activity/function may be measured according to the test methods described in Example 9 or 20
  • the invention relates to nucleic acid constructs comprising a nucleotide sequence encoding a CART polypeptide or a fragment or variant thereof with appetite regulating activity/function.
  • the invention relates to nucleic acid constructs encoding a polypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to 9 such as the sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are linked by disulphide bonds in the configuration I-III, II-V and IV-VI when the cysteines are numbered from the N-terminal end.
  • the invention relates to recombinant vectors comprising the nucleic acid constructs and recombinant host cells comprising the nucleic acid constructs or the vectors.
  • the invention relates to a method of producing a CART polypeptide or a fragment or a variant thereof with appetite regulating activity /function which method comprises cultivating a host cell as defined above in a suitable culture medium under conditions permitting expression of the nucleic acid construct and recovering the resulting polypeptide from the culture medium/cell.
  • the invention relates to transgenic animals or transgenic plants comprising the nucleic acid construct as defined above as well as methods of producing a CART polypeptide or a fragment or a variant thereof with appetite regulating activity/function using such transgenic animals or transgenic plants.
  • the invention relates to an antibody capable of specifically binding to a CART polypeptide or a fragment or a variant thereof with appetite regulating activity/function, such as a polypeptide with a sequence selected from the sequences SEQ ID Nos. 1 to 9, e.g. the sequences SEQ ID Nos. 1 to 9 in which the cysteine residues are linked by disulphide bonds in the configuration I-III, II-V and IV-VI when the cysteines are numbered from the N-terminal end.
  • the antibody is monoclonal and the invention furthermore relates to hybridomas producing such monoclonal antibodies.
  • the invention relates to appetite regulating compositions comprising the polypeptides as defined above and a pharmaceutically acceptable carrier and the use of the polypeptides for the preparation of medicaments for the regulation of appetite.
  • the medicaments are used for the treatment of obesity.
  • the invention relates to a method for the regulation of appetite comprising administering to a subject in need thereof an effective amount of a polypeptide as defined above.
  • the invention relates to the use of CART or CART fragments or variants to identify a functional receptor and the subsequent use of the CART receptor to identify receptor agonists with appetite regulating activity.
  • the invention relates to compounds that upregulate the CART expression and thereby regulate appetite.
  • Figure 2 Expression in E. coli. Fusion to Glutathione-S-transferase.
  • Figure 3 Expression in E. coli. Fusion to Thioredoxin.
  • Figure 4 Expression of CART in yeast.
  • Figure 5 Sequence of heterologous protein expression cassette of plasmid pEA182 and pEA183 in yeast.
  • FIG. 7 Analytical HPLC of CART yeast supernatant H-372.
  • Figure 8 SP-Sepharose Column.
  • Figure 9 Analytical HPLC of CART fragment, pool A.
  • Figure 10 Analytical HPLC of CART fragment, pool B.
  • Figure 11 Analytical HPLC of CART fragment, pool C.
  • Figure 12 Primary and secondary structure of "IPI-CART" showing the I-III, II-V and IV- VI disulphide bond configuration.
  • Figure 13 Effect of fasting on CART mRNA expression.
  • Figure 14 CART mRNA in Zucker rat arcuate nucleus and heterozygote controls.
  • Figure 15 S. cerevisiae plasmid for the expression and secretion of Glu-Glu-Ile-Asp- CART(55-102).
  • TPI-prom. and TPI-term. are S. cerevisiae triosephosphate isomerase transcription promoter and terminator sequences, respectively.
  • TPI S. pombe is the Schizosaccharmyces pombe triosephosphate isomerase gene. Only restriction sites relevant for the plasmid construction have been indicated.
  • This invention is based on the unexpected and surprising discovery that CART polypeptide has been found to possess appetite regulating function/activity.
  • polypeptide is understood to include a mature protein or a precursor form thereof as well as a functional fragment thereof which essentially has the activity of the full-length polypeptide.
  • polypeptide is intended to include homologues of said polypeptide.
  • homologues comprise an amino acid sequence exhibiting a degree of identity of at least
  • the homologue of the polypeptide may be one encoded by a nucleotide sequence hybridizing with an oligonucleotide probe prepared on the basis of the polypeptide sequences shown in SEQ ID Nos. 1-9 .
  • the invention relates to a variant of the polypeptide of the invention.
  • the variant is one in which one or more amino acid residues in one or more positions have been substituted by other amino acid residues.
  • Homologues of the present polypeptide may have one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids, small amino- or carboyxyl- terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly- histidine tract, an antigenic epitope or a binding domain. See in general Ford et al. , Protein Expression and Purification 2: 95-107, 1991.
  • conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).
  • basic amino acids such as arginine, lysine, histidine
  • acidic amino acids such as glutamic acid and aspartic acid
  • polar amino acids such as glutamine and asparagine
  • hydrophobic amino acids such as leucine, isoleucine, valine
  • aromatic amino acids such as phenylalanine, tryptophan, tyrosine
  • small amino acids such as glycine, alanine, serine,
  • amino acids essential to the activity of the polypeptide of the invention may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244. 1081-1085, 1989). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. appetite regulation) to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffmity labelling. See, for example, de Vos et al., Science 255: 306-312, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al. , FEBS Lett. 309: 59-64, 1992.
  • the homologue may be an allelic variant, i.e. an alternative form of a gene that arises through mutation, or an altered polypeptide encoded by the mutated gene, but having substantially the same activity as the polypeptide of the invention. Hence mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • the homologue of the present polypeptide may also be a species homologue, i.e. a polypeptide with a similar activity derived from another mammalian species eg. rat, mouse, sheep or human.
  • homologues of said polypeptide may be found in other tisssues such as the brain and pancreas.
  • a homologue of the polypeptide may be isolated by preparing a genomic or cDNA library of a cell of the species or tissue in question, and screening for DNA sequences coding for all or part of the homologue by using synthetic oligonucleotide probes in accordance with standard techniques, e.g. as described by Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, or by means of polymerase chain reaction (PCR) using specific primers as described by Sambrook et al. and Saiki at al. , Science 239 (1988) 487-491.
  • standard techniques e.g. as described by Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, or by means of polymerase chain reaction (PCR) using specific primers as described by Sambrook et al. and Saiki at al. , Science 239 (1988) 487-491.
  • polypeptide in a highly purified form , i.e. greater than 90% pure, more preferably 95% and most preferably 99% pure, as determined by analytical HPLC.
  • the currently preferred polypeptides of the invention are the ones comprising the amino acid sequences shown in SEQ ID Nos. 1-9.
  • nucleic acid construct is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin.
  • construct is intended to indicate a nucleic acid segment which may be single or double stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The construct may optionally contain other nucleic acid segments.
  • the nucleic acid construct of the invention encoding the polypeptide of the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., supra).
  • the DNA sequence encoding the polypeptide is preferably of mammalian origin, i.e. derived from a genomic DNA or cDNA library. More preferably, the DNA sequence may be of rodent origin, e.g. rat or mice origin. Even more preferably, the DNA sequence may be of human origin.
  • the nucleic acid construct of the invention encoding the polypeptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.
  • the nucleic acid construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al. , Science 239 (1988), 487 - 491.
  • primers for instance as described in US 4,683,202 or Saiki et al. , Science 239 (1988), 487 - 491.
  • As template for the PCR cloning we used the same double stranded cDNA preparation as described in example 1 (from MSL-A-AN). The PCR reaction, 25 cycles: 60 sec 94°C
  • the nucleic acid construct is preferably a DNA construct which term will be used exclusively in the following.
  • the present invention relates to a recombinant vector comprising a DNA construct of the invention.
  • the recombinant vector into which the DNA construct of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the polypeptide of the invention is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA encoding the polypeptide of the invention in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809 - 814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (US 4,745,051; Vasuvedan et al., FEBS Lett. 311. (1992) 7 - 11), the P10 promoter (J.M. Vlak et al., J. Gen. Virology 69. 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (US 5, 155,037; US 5, 162,222), or the baculovirus 39K delayed-early gene promoter (US 5,155,037; US 5,162,222).
  • promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al.. J. Biol. Chem. 255 (1980). 12073 - 12080; Alber and Kawasaki, J. Mol. Appl. Gen. I (1982), 419 - 434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652 - 654) promoters.
  • suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093 - 2099) or the tpjA promoter.
  • suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • Preferred are the TAKA -amylase and gluA promoters.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus subtilis xylosidase gene, or by the phage Lambda P R or P L promoters or the E. coli lac, trp or tac promoters.
  • the DNA sequence encoding the polypeptide of the invention may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., OJ . cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, or cit.) or ADH3 (McKnight et al., or cit.) terminators.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV40 origin of replication.
  • suitable sequences enabling the vector to replicate are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • sequences enabling the vector to replicate are e.g. the Col El origin of replication as in pUC19 or pBR322 or the pl5A origin of replication as in pACYC184 when the bacterium is E. coli.
  • the origin of replication from e.g. pUBHO is often used.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P.R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate.
  • selectable markers include arndS, pyrG. argB, niaD. sC .
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the polypeptide in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide.
  • the secretory signal sequence may be that normally associated with the polypeptide or may be from a gene encoding another secreted protein.
  • the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed polypeptide into the secretory pathway of the cell.
  • the signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the ⁇ -factor signal peptide (cf. US 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al.. Nature 289. 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L.A. Vails et al., £ejj 4J£, 1987, pp.
  • yeast BARl signal peptide cf. WO 87/02670
  • yeast aspartic protease 3 YAP3
  • a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the polypeptide.
  • the function of the leader peptide is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell).
  • the leader peptide may be the yeast ⁇ -factor leader (the use of which is described in e.g.
  • the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.
  • the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, or a gene encoding a Humicola lanuginosa lipase.
  • the signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral ⁇ - amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
  • the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. US 5,023,328).
  • the DNA sequence encoding the present polypeptide introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/ or terminator sequence than in its natural environment.
  • the term "homologous” is intended to include a cDNA sequence encoding a polypeptide native to the host organism in question.
  • heterologous is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present polypeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • Examples of bacterial host cells which, on cultivation, are capable of producing the polypeptide of the invention are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis , or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as Echerichia coli.
  • the transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the polypeptide When expressing the polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.
  • sonication or osmotic shock to release the contents of the periplasmic space and recovering the polypeptide.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines.
  • Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601 - 621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982). 327 - 341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982). 422 - 426; Wigler et al.
  • yeasts cells examples include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or
  • Saccharomyces kluyveri Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are described, e.g. in US 4,599,311, US 4,931,373, US 4,870,008, US 5,037,743, and US 4,845,075, all of which are hereby incorporated by reference.
  • Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine.
  • a preferred vector for use in yeast is the POT1 vector disclosed in US 4,931,373.
  • the DNA sequence encoding the polypeptide of the invention may be preceded by a signal sequence and optionally a leader sequence , e.g.
  • yeast cells are strains of Kluyveromyces , such as K. lactis, Hansenula, e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., Gen. Microbiol. 132. 1986, pp. 3459-3465; US 4,882,279).
  • Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A. oryzae, A. nidulans or A. niger.
  • Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277 and EP 230 023.
  • the transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al. , 1989, Gene 78: 147-156.
  • a filamentous fungus When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct of the invention, conveniently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell.
  • This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.
  • Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in US 4,745,051; US 4,879,236; US 5,155,037; 5,162,222; EP 397,485; all of which are incorporated herein by reference.
  • the insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. US 5,077,214).
  • Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.
  • the transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the present polypeptide, after which the resulting polypeptide is recovered from the culture.
  • the medium used for culturing the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the polypeptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
  • a salt e.g. ammonium
  • transgenic animal technology to produce the present polypeptide.
  • a transgenic animal is one in whose genome a heterologous DNA sequence has been introduced.
  • the polypeptide of the invention may be expressed in the mammary glands of a non-human female mammal, in particular one which is known to produce large quantities of milk. Examples of preferred mammals are livestock animals such as goats, sheep and cattle, although smaller mammals such as mice, rabbits or rats may also be employed.
  • the DNA sequence encoding the present polypeptide may be introduced into the animal by any one of the methods previously described for the purpose. For instance, to obtain expression in a mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include the genes encoding casein (cf. US 5,304,489), beta- lactoglobulin, alpha-lactalbumin and whey acidic protein. The currently preferred promoter is the beta-lactoglobulin promoter (cf. Whitelaw et al., Biochem J. 286, 1992, pp. 31-39). It is generally recognized in the art that DNA sequences lacking introns are poorly expressed in transgenic animals in comparison with those containing introns (cf .
  • One such region is a DNA segment which provides for intron splicing and RNA polyadenylation from the 3' non-coding region of the ovine beta-lactogloblin gene. When substituted for the native 3' non-coding sequences of a gene, this segment will enhance and stabilise expression levels of the polypeptide of interest. It may also be possible to replace the region surrounding the initiation codon of the polypeptide of interest with corresponding sequences of a milk protein gene. Such replacement provides a putative tissue-specific initiation environment to enhance expression.
  • a nucleotide sequence encoding the polypeptide is operably linked to additional DNA sequences required for its expression to produce expression units.
  • additional sequences include a promoter as indicated above, as well as sequences providing for termination of transcription and polyadenylation of mRNA.
  • the expression unit further includes a DNA sequence encoding a secretory signal sequence operably linked to the sequence encoding the polypeptide.
  • the secretory signal sequence may be one native to the polypeptide or may be that of another protein such as a milk protein (cf. von Heijne et al., Nucl. Acids Res. 14, 1986, pp. 4683- 4690; and US 4,873,316).
  • Construction of the expression unit for use in transgenic animals may conveniently be done by inserting a DNA sequence encoding the present polypeptide into a vector containing the additional DNA sequences, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA sequence encoding a milk protein and to replace the coding region for the milk protein with a DNA sequence coding for the present polypeptide, thereby creating a fusion which i innpcllnuHdesc p evxpresccs ⁇ i ⁇ on C sAe ⁇ qHuPeTnIcPeOs t thhje» m miilllk"" n prrnott ⁇ eiin gtetnie.
  • the expression unit is then introduced into fertilized ova or early-stage embryos of the selected host species.
  • Introduction of heterologous DNA may be carried out in a number of ways, including microinjection (cf. US 4,873,191), retroviral infection (cf. Jaenisch,
  • Production in transgenic plants may also be employed.
  • WO 89/12386 discloses the transformation of plant cells with a gene coding for levansucrase or dextransucrase, regeneration of the plant (especially a tomato plant) from the cell resulting in fruit products with altered viscosity characteristics.
  • the DNA sequence encoding the present polypeptide is under the control of a regulatory sequence which directs the expression of the polypeptide from the DNA sequence in plant cells and intact plants.
  • the regulatory sequence may be either endogenous or heterologous to the host plant cell.
  • the regulatory sequence may comprise a promoter capable of directing the transcription of the DNA sequence encoding the polypeptide in plants.
  • promoters capable of directing the transcription of the DNA sequence encoding the polypeptide in plants.
  • promoters which may be used according to the invention are the 35s RNA promoter from cauliflower mosaic virus (CaMV), the class I patatin gene B 33 promoter, the ST-LSl gene promoter, promoters conferring seed-specific expression, e.g. the phaseolin promoter, or promoters which are activated on wounding, such as the promoter of the proteinase inhibitor II gene or the wunl or wun2 genes.
  • the promoter may be operably connected to an enhancer sequence, the purpose of which is to ensure increased transcription of the DNA sequence encoding the polypeptide.
  • enhancer sequences are enhancers from the 5 '-upstream region of the 35s RNA of CaMV, the 5 '-upstream region of the ST-LSl gene, the 5 '-upstream region of the Cab gene from wheat, the 5 '-upstream region of the 1'- and 2 '-genes of the TR-DNA of the Ti plasmid pTi ACH5, the 5 '-upstream region of the octopine synthase gene, the 5 '-upstream region of the leghemoglobin gene, etc.
  • the regulatory sequence may also comprise a terminator capable of terminating the transcription of the DNA sequence encoding the polypeptide in plants.
  • suitable terminators are the terminator of the octopine synthase gene of the T-DNA of the Ti- plasmid pTiACH5 of A robacterium tumefaciens. of the gene 7 of the T-DNA of the Ti plasmid pTiACH5, of the nopaline synthase gene, of the 35s RNA-coding gene from CaMV or from various plant genes, e.g. the ST-LSl gene, the Cab gene from wheat, class I and class II patatin genes, etc.
  • the DNA sequence encoding the polypeptide may also be operably connected to a DNA sequence encoding a leader peptide capable of directing the transport of the expressed polypeptide to a specific cellular compartment (e.g. vacuoles) or to extracellular space.
  • suitable leader peptides are the leader peptide of proteinase inhibitor II from potato, the leader peptide and an additional about 100 amino acid fragments of patatin, or the transit peptide of various nucleus-encoded proteins directed into chloroplasts (e.g. from the St-LSl gene, SS-Rubisco genes, etc.) or into mitochondria (e.g. from the ADP/ATP translocator).
  • DNA sequence encoding the polypeptide may be modified in the 5' non- translated region resulting in enhanced translation of the sequence. Such modifications may, for instance, result in removal of hairpin loops in RNA of the 5' non-translated region.
  • Translation enhancement may be provided by suitably modifying the omega sequence of tobacco mosaic virus or the leaders of other plant viruses (e.g. BMV, MSV) or of plant genes expressed at high levels (e.g. SS-Rubisco, class I patatin or proteinase inhibitor II genes from potato) .
  • the DNA sequence encoding the polypeptide may furthermore be connected to a second DNA sequence encoding another polypeptide or a fragment thereof in such a way that expression of said DNA sequences results in the production of a fusion protein.
  • the second DNA sequence may, for instance, encode patatin or a fragment thereof (such as a fragment of about 100 amino acids).
  • the plant in which the DNA sequence coding for the polypeptide is introduced may suitably be a dicotyledonous plant, examples of which are is a tobacco, potato, tomato, or leguminous (e.g. bean, pea, soy, alfalfa) plant. It is, however, contemplated that mono- cotyledonous plants, e.g. cereals, may equally well be transformed with the DNA sequence coding for the enzyme.
  • E. coli Procedures for the genetic manipulation of monocotyledonous and dicotyledonous plants are well known.
  • numerous cloning vectors are available which generally contain a replication system for E. coli and a selectable/screenable marker system permitting the recognition of transformed cells.
  • These vectors include e.g. pBR322, the pUC series, pACYC, M13 mp series etc.
  • the foreign sequence may be cloned into appropriate restriction sites.
  • the recombinant plasmid obtained in this way may subsequently be used for the transformation of E. coli.
  • Transformed E. coli cells may be grown in an appropriate medium, harvested and lysed.
  • the chimeric plasmid may then be reisolated and analyzed. Analysis of the recombinant plasmid may be performed by e.g. determination of the nucleotide sequence, restriction analysis, electrophoresis and other molecular-biochemical methods. After each manipulation the sequence may be cleaved and ligated to another DNA sequence. Each DNA sequence can be cloned on a separate plasmid DNA. Depending on the way used for transferring the foreign DNA into plant cells other DNA sequences might be of importance.
  • the Ti-plasmid or the Ri plasmid of Agrobacterium tumefaciens or Agrobacterium rhizogenes at least the right border of the T-DNA may be used, and often both the right and the left borders of the T-DNA of the Ri or Ti plasmid will be present flanking the DNA sequence to be transferred into plant cells.
  • T-DNA for transferring foreign DNA into plant cells
  • this sequence is fairly stable at the original locus and is usually not lost in subsequent mitotic or meiotic divisions.
  • a selectable marker gene will be cotransferred in addition to the gene to be transferred, which marker renders the plant cell resistant to certain antibiotics, e.g. kanamycin, hygromycin, G418 etc. This marker permits the recognition of the transformed cells containing the DNA sequence to be transferred compared to nontransformed cells. Numerous techniques are available for the introduction of DNA into a plant cell.
  • Examples are the Agrobacterium mediated transfer, the fusion of protoplasts with liposomes containing the respective DNA, microinjection of foreign DNA, electroporation etc.
  • the DNA to be transferred has to be present in special plasmids which are either of the intermediate type or the binary type. Due to the presence of sequences homologous to T-DNA sequences, intermediate vectors may integrate into the Ri- or Ti-plasmid by homologous recombination. The Ri- or Ti-plasmid additionally contains the vir-region which is necessary for the transfer of the foreign gene into plant cells.
  • Binary vectors may replicate in both Agrobacterium species and E. coli. They may contain a selectable marker and a poly-linker region which to the left and right contains the border sequences of the T-DNA of Agrobacterium rhizogenes or Agrobacterium tumefaciens. Such vectors may be transformed directly into Agrobacterium species.
  • the Agrobacterium cell serving as the host cell has to contain a vir-region on another plasmid. Additional T-DNA sequences may also be contained in the Agrobacterium cell.
  • the Agrobacterium cell containing the DNA sequences to be transferred into plant cells either on a binary vector or in the form of a cointegrate between the intermediate vector and the T-DNA region may then be used for transforming plant cells.
  • multicellular explants e.g. leaf discs, stem segments, roots
  • single cells protoplasts
  • cell suspensions are cocultivated with Agrobacterium cells containing the DNA sequence to be transferred into plant cells.
  • the plant cells treated with the Agrobacterium cells are then selected for the cotransferred resistance marker (e.g. kanamycin) and subsequently regenerated to intact plants. These regenerated plants will then be tested for the presence of the DNA sequences to be transferred.
  • cotransferred resistance marker e.g. kanamycin
  • DNA is transferred by e.g. electroporation or microinjection, no special requirements are needed to effect transformation.
  • Simple plasmids e.g. of the pUC series may be used to transform plant cells.
  • Regenerated transgenic plants may be grown normally in a greenhouse or under other conditions. They should display a new phenotype (e.g. production of new proteins) due to the transfer of the foreign gene(s).
  • the transgenic plants may be crossed with other plants which may either be wild-type or transgenic plants transformed with the same or another DNA sequence. Seeds obtained from transgenic plants should be tested to assure that the new genetic trait is inherited in a stable Mendelian fashion.
  • the present polypeptide may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences. 19 th. edition, 1995.
  • the composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution.
  • the compositions may be sterilized by conventional sterilization techniques which are well known in the art.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the pharmaceutical composition of the present invention may also be adapted for oral, nasal, transdermal, transepithelial or rectal administration.
  • the pharmaceutically acceptable carrier or diluent employed in the composition may be any conventional solid carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • the composition may be tabletted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge.
  • the amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.
  • the present polypeptide may also be placed in a soft gelatin capsule in a liquid carrier such as syrup, peanut oil, olive oil or water.
  • the polypeptides of the invention are effective over a wide dosage range.
  • a typical dosage is in the range of from 0.05 to about 1000 mg, preferably from about 0.1 to about 500 mg, and more preferred from about 0.5 mg to about 200 mg per day administered in one or more dosages such as 1 to 3 dosages.
  • the exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated as well as other factors evident to those skilled in the art.
  • the polypeptide of the invention is contemplated to be advantageous for use in therapeutic applications within appetite suppression or satiety induction, such as for the prophylaxis or treatment of diseases or disorders associated with impaired appetite regulation. Examples of such diseases or disorders are obesity, type II diabetes and bulimia.
  • the dosage of the polypeptide administered to a patient will vary with the type and severity of the condition to be treated, but is generally in the range of 0.01-5.0 mg/kg body weight per day in one or more dosages such as 1 to 3 dosages.
  • polypeptide of the invention is contemplated to be advantageous for treatment of autoimmune disorders, inflammation, arthritis, type I diabetes, multiple schlerosis, stroke, osteoporosis, septic shock, symptoms of menopause, menstrual complications and Parkinson's disease.
  • RNA from cultivated cells were prepared from primary cell lines derived from the rat tumors MSL-A-AN and MSL-A-M3 (Madsen et al, Scand. J. Clin. Invest. Supplement 220: 27-36) by the method of Chomczynski & Sacchi (1987). From this we made poly-A mRNA using Pharmacia's "Quick Prep® micro " mRNA purification kit. Double stranded cDNA was made using Clontech's "cDNA clone I" synthesis kit. Eco RI adaptors CA6/CA7 (Ace et al. (1994)) were added to the blunt ended cDNA's. 50 ng of each cDNA was amplified using the CA6 primer. The primer used for amplification of the MSL-A-M3 cDNA was biotinylated (the driver). The amplified MSL- A-AN cDNA was cut with Eco RI (the tracer).
  • the cells were plated on LB plates with 100 ⁇ g/ml Ampicillin. The plates were replicated and the cells from the replica were transferred to nylon filters (Hybond-N + , Amersham). The filters were hybridized with radioactively labelled driver cDNA and autoradiographs were made. The filters were then stripped for the radioactive probe and thereafter the procedure was repeated with a probe made from the tracer cDNA. The autoradiographs were compared and spots that were only present with the latter probe were identified and the corresponding colonies were isolated.
  • DNA sequencing of the inserted DNA in one of these colonies were identified as CART (Cocaine and Amphetamine Regulated Transcript from rat brain (Douglass et al. (1995)).
  • This transcript codes for a protein (polypeptide) of 129 or 116 amino acids (differential splicing of an in frame 39 bp intron).
  • the polypeptide seems to have a signal sequence in the amino terminal end, and the secreted part contains several dibasic amino acid pairs which could be "pro hormone" processing sites.
  • reaction mixtures were cut with Hin dill, filled out with Klenow polymerase, and then cut with Bam HI. They were then run on a 2% agarose gel and the bands corresponding to the three variants were isolated and cloned into pGEX-2T (cut with Eco RI, filled out, and then cut with Bam HI) giving rise to pSX600 (IPI-CART) corresponding to SEQ ID No. 4, pSX 601 (short form) corresponding to SEQ ID No. 2, and pSX605 (long form) corresponding to SEQ ID No. 1 (see Fig. 2).
  • reaction mixtures were cut with Hin dill, filled out with Klenow polymerase and then cut with Bam HI. They were then run on a 2% agarose gel and the bands corresponding to the three variants were isolated and cloned into pTrxFus (cut with Sal I, filled out, and then cut with Bam HI) giving rise to pSX610 (long form) corresponding to SEQ ID No. 1, pSX ⁇ ll (short form) corresponding to SEQ ID No. 2, and pSX612 (IPI-CART) corresponding to SEQ ID No. 4 (see Fig. 3).
  • the plasmids were transformed into E. Coli G1724 (Invitrogen) and the the resulting strains were cultivated according to the manual for the ThioFusionTM Expression System kit.
  • the Yeast-E.coli shuttle vector used in the following example contains a heterologous protein expression cassette, which includes a DNA sequence encoding a modified MF ⁇ l leader sequence (with a Ncol site added in the 3 '-end) followed by the heterologous polypeptide in question operably placed in between the TPI promoter and the
  • CART1 5'-ACG GAG GAG CCC ATG GCT AAG AGA CAG GAG GAT GCC
  • PCR Polymerase Chain Reaction
  • the resulting PCR products were cut with restriction enzymes Ncol and Xbal and ligated with the BstXI/Xbal fragment and the BstXI/NcoI fragment of pAK405.
  • BstXI, Ncol and Xbal cutat positions 701, 1419 and 1616 of pAK405, respectively.
  • the construction and DNA sequence of the resulting heterologous expression cassettes are shown in figures 4 and 5, respectively.
  • pEA182 short form of CART
  • pEA183 long form of CART
  • S. cerevisia strain ME1487 MAT ⁇ ⁇ yap3::URA3 pep4-3 ⁇ tpi::LEU2 leu2 HIS4 ⁇ URA3, described in patent application DK 0749/96.
  • Transformants were selected by glucose utilization as a carbon source in YPD plates (1 % w/v yeast extract, 2% w/v peptone, 2% glucose, 2% agar).
  • yEA182 corresponding to SEQ ID No. 2 and yEA183 corresponding to SEQ ID No. 1 are the transformants obtained from the plasmids pEA182 and pEA183, respectively.
  • IPI-CART pSX630 corresponding to SEQ ID No. 4.
  • Transformants were cultivated in YPD liquid medium at 30°C for 3 days with shaking at 200 rpm. Culture supernatants were obtained after centrifugation and supernatants were analysed for CART related material.
  • Human CART(l-89) corresponding to SEQ ID No. 3 differs from the rat form by having a valine residue in position 42 in stead of a isoleucine residue.
  • Human CART(l-89) may be prepared in analogy with the above examples starting from a human tissue or simply by substituting valine for isoleucine in position 42 of rat CART(l-89) according to methods well-known to a person skilled in the art.
  • Example 7 Ten mg of rat CART(54-102, long), SEQ ID No.6, prepared as described in Example 7, was dissolved in 2 ml of 70% (v/v) formic acid. A crystal corresponding to approx. 1 mg of cyanogenebromide was added to the dissolved peptide and the mixture was allowed to stand dark at room temperature for 16 hours. The generated CART fragment, CART(68- 102, long) was purified by preparative HPLC as described in Example 7.
  • the thioredoxin-CART short form fusion protein was isolated from 2 litres of E.Coli fermentation broth and subjected to FXa cleavage.
  • Fraction 15 The peptide found in Fraction 15 is identical to rat CART( 10-89) corresponding to SEQ ID No. 10, whereas the peptide in Fraction 28 is the thioredoxin split product with the C- terminal sequence of ...IEGR.
  • the small peptide fragment, CART(l-9) was not identified in the digest. From 2 1 of fermentation broth the total of 4.0 mg of CART( 10-89) was isolated.
  • Human CART(10-89) corresponding to SEQ ID No. 11 differs from the rat form by having a valine residue in position 33 in stead of a isoleucine residue.
  • Human CART(10-89) may be prepared in analogy with the above example starting from a human tissue or simply by substituting valine for isoleucine in position 33 of rat CART(10-89) according to methods well-known to a person skilled in the art.
  • Yeast construction The fermentation broth from the 5 litres yeast fermentation (yEA183, long form) was analysed by HPLC (Fig. 7). A series of expression products was seen in this analysis. Preliminary sequence analysis indicated that several of the peptides eluting at a retention time between 20 and 30 min. were fragments of the mature full length CART molecule. The total amount of CART related products in the fermentation broth was approx. 250 mg/litre. The CART fragments from 4.25 litres of fermentation broth were separated using the following method:
  • the column was eluted with a linear gradient between 1500 ml of 50 mM HAc and 1500 ml of 50 mM HAc containing 1.0 M NaCl. Fractions of 10 ml were collected and analysed for the content of CART fragments by analytical HPLC. The chromatogram from the ion exchange chromatography is shown in Fig. 8.
  • Three pools were generated on the basis of the analytical HPLC analysis of the individual fractions. Each of these pools, representing a well defined CART fragment, were further purified by preparative HPLC.
  • the individual pools 120 - 150 ml were pumped on a Vydac 214TP1022 (100ml) reverse phase C4 HPLC column previously equilibrated with 0.1 % TFA. The column was washed with 100 ml 0.1 % TFA and eluted with a linear gradient from 0 to 70% MeCN in 0.1 % TFA at a flow rate of 3 ml/min.
  • the individual fractions from the 3 preparative HPLC purifications were analysed by analytical HPLC and the 3 individual CART fragments were isolated from these fractions by lyophilisation.
  • CART(62-102) corresponds to SEQ ID No. 8
  • CART(61-102) corresponds to SEQ ID No. 7
  • CART(54-102) corresponds to SEQ ID No. 6.
  • Human CART(62-102) and CART(61-102) fragments are identical to the rat fragments.
  • Human CART(54-102) differs from the rat fragment by having a valine residue in position 2 in stead of a isoleucine residue.
  • Human CART(54-102) may be prepared using the same method as described for rat CART(54-102) starting from a human tissue or simply by substituting valine for isoleucine in position 2 of rat CART(54-102) according to methods well-known to a person skilled in the art.
  • SEQ ID No. 8 The C-terminal part of the CART molecule contains 6 cysteine residues: Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val- Arg-Lys-Gly-Ala-Arg-lie-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly- Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu
  • the CART fragment (residues 62-102) prepared as described in the preceding example was digested with Armillaria Mellea protease, which cleaves specifically on the N-terminal side of lysine residues.
  • the fragments generated were separated by HPLC and subjected to mass spectrometry and amino acid sequence analyses. From mass spectrometry and sequence analysis it could be deduced that the following two fragments were generated by the
  • the first of these fragments is a 3 chained molecule still held together by the 3 disulphide bonds. This molecule was subjected to digestion with Pseudomonas fragi endoproteinase Asp-N, which cleaves specifically on the N-terminal side of aspartic acid residues. From this digest the following two fragments could be isolated:
  • the first of these fragments is a two chained molecule held together by a single disulphide bond.
  • cysteine residue I and III of the original molecule must be linked.
  • the second of these fragments, containing cysteine residue II, IV, V and VI, is a 3 chained molecule linked by 2 disulphide bonds. This molecule was subjected to trypsin digestion and the following two fragments were generated:
  • mice were deprived of their normal feed for two days and given free access to a solution of nutritionally complete infant formula milk (Complan ® ) for the first day, after which food deprivation was complete for the last day before testing.
  • mice were injected intra-cerebroventricularly (ICV) in the lateral ventricle with 10 microlitres of a solution containing the test substance.
  • ICV intra-cerebroventricularly
  • the drinking tube was connected to a reservoir containing the formula milk solution, and the interior of the drinking tube contained an electrode enabling the detection of drinking contacts with the solution by measuring the flow of a weak (unnoticeable) electric current through mice by means of an electronic apparatus connected to the drinking tube electrode and the stainless steel grid floor. Consumption of the milk solution was measured over a 10 minutes period by electronically recording the total amount of contact with the milk solution during the test session.
  • the degree of appetite suppression produced by a given test substance was determined by statistical comparison of the duration of milk consumption by control (vehicle treated) mice with that of mice treated with a test substance. The degree of appetite suppression in a treated group of mice was expressed as percent reduction of consumption relative to the mean of the control group's response.
  • mice were tested for appetite suppression as described in Example 9 after treatment with 0.5- 10 micrograms of a test substance consisting of recombinant rat CART(54-102) dissolved in phosphate buffered saline. Intra-cerebro ventricular injections of the test substance produced statistically significant suppression of milk consumption.
  • mice were tested for appetite suppression as described in Example 9 after treatment with 0.5- 10 micrograms of a test substance consisting of recombinant rat CART(62-102) dissolved in phosphate buffered saline. Intra-cerebroventricular injections of the test substance produced statistically significant suppression of milk consumption.
  • Rat brain tissue was obtained from two groups of Zucker rats (6 animals in each group): fa/fa and fa/ + .
  • Cryostat sections were cut and three sections were used for in situ hybridisation with 35-S labelled anti-sense CART RNA (rCART5A cDNA (Eco47-HindIII fragment from bp Nos. 226-411)).
  • Post-hybridisation washings were performed at 57°C and 62 °C in 50 % formamide. Additional sections were included with a similar amount of 35-S labelled sense RNA probe and these showed no signal.
  • Slides were exposed on one Betamax hyperfilm for 12 days.
  • the images were digitized to 256 grey levels and analysed using the NIH Image software (treatment blinded to the observer).
  • An empirically determined gray level (100) was used to set the threshold on all images after exclusion of those with bad morphology or bad representation of the area in question (three sections) and one set of slides (Nos. 25-27) as one animal due to an error was represented twice (and one missing).
  • the average gray scale value of all pixels above the arcuate nucleus was then measured. A mean for each animal was then determined and the product of the area and mean calculated.
  • Plasmid pSX637 encoding Glu-Glu-Ile-Asp-CART(55-102), was constructed by the use of the PCR technique "Splicing by Overlap Extension” (Horton et al. , Gene 77:61-68, 1989) and the product was inserted into pAK405 (Example 5).
  • the resulting expression plasmid (pSX637) is shown in Fig.15.
  • a sequence of: Lys-Glu-Leu- Glu has been placed between the ⁇ -leader and Kex2 site in order to optimise processing (Kjeldsen et al., Gene 170: 107-112, 1996).
  • Plasmid pSX637 was transformed into Saccharomyces. cerevisiae strains ME1487 ⁇ MATa Ayap3::URA3 Atpi::LEU2 pep4-3 Aura3 leu2) and ME1719 (MATa I MATa
  • Host cells were cultured in YPGGE medium (1 %(w/v) yeast extract, 2%(w/v) peptone, 2%(w/v) galactose, 2%(v/v) glycerol and l %(v/v) ethanol) to ODgoo nm of 0.2. Transformation was made by using a standard protoplast method.
  • Transformant YES1789 was obtained containing the EEID-CART expressing plasmid after transformants were selected on minimal plates containing glucose.
  • Fermentation of EEID-CART(55-102) (yeast strain: YES1789) was carried out in 6 L stainless steel fermentor from Chemap A/B, Switzerland equipped with bottom stirrer and in situ steam sterilization. The medium was composed essentially as previously described (Thim et al. , FEBS Lett. 318:345-352, 1993) and a starting volume of 4.0 L was chosen. Ammonia was added to adjust pH throughout the fermentation to 4.9 and the temperature was kept constant at 30 °C with steam/cooling water. Dissolved oxygen was kept above 50% saturation by frequent adjustment of the stirrer speed.
  • the inoculum was from a YPD (Yeast-extract Peptone Dextrose) culture (2 days, 30 °C).
  • Glucose (1250 g) was dissolved in water to a volume of 2 L, sterilised separately in an autoclave (30 min, 121 °C), and added with a constant rate of 30 g/h over the first 24 hours. The rate was increased to 60 g/h over the next 24 hours. After 48 hours of cultivation the broth was harvested.
  • the EEID-CART(55-102) broth was adjusted to pH 11 with 3 N NaOH and kept at 25°C for 30 min before centrifugation as above.
  • the supernatant was adjusted to pH 9.7 to protect against proteolysis before the purification was initiated.
  • the dry biomass in the two fermentations was 73.6 g/L.
  • the weight of the total fermentation broth was 5706 g.
  • the fermentation supernatant (4.6 L) from yeast strain YES 1789 expressing Glu-Glu-Ile- Asp-CART(55-102) was dialysed against 60 L of water at 4°C for 96 h.
  • the pH was adjusted to 4.3 and the solution was pumped onto a SP-Sepharose (Pharmacia) column (5 x 15 cm) with a flow rate of 300 mL/h. Prior to the application the column was equilibrated with 50 mM HAc buffer pH 4.25. The column was washed with 3 L of 50 mM HAc buffer pH 4.25. EEID-CART(55-102) was eluted from the column by a linear gradient between 1.5 L of 50 mM HAc buffer pH 4.25 and 1.5 L of 50 mM HAc buffer pH 4.25 containing IM NaCl. Fractions (10 mL) were collected at a flow rate of 100 mL/h and the absorbance was measured at 280 nm.
  • the EEID-CART(55-102) molecule eluted at 0.5M of NaCl and fractions containing the peptide were dialysed against 25 L of 50 mM HAc buffer pH 4.5 at 4°C for 96 h.
  • L-Cystein was added to the solution (560 mL) to give a final concentration of 1 mM, and 4.5 mL dipeptidylaminopeptidase-1 ( DAP-1, Cathepsin C from chicken liver, EC 3.4.14.1, Unizyme Laboratories) was added.
  • DAP-1 dipeptidylaminopeptidase-1
  • the column was washed with 1.6 L of equilibration buffer and CART(55-102) was eluted with a linear gradient between 1 L of 50 mM HAc buffer pH 4.25 and 1 L of 50 mM HAc buffer pH 4.25 containing 1.2 M NaCl, at a flow rate of 100 mL/h.
  • the absorbance at 280 nm was recorded and fractions of 10 mL were collected.
  • the CART(55-102) molecule eluted at 0.67 M of NaCl and fractions containing the peptide were pooled. The solution was divided into 5 equal portions.
  • the total yield of CART(55-102) from 4.6 L of fermentation supernatant was 705 mg.
  • N-terminal amino acid sequences were determined by automated Edman degradations using an Applied Biosystem Model 494 Protein Sequencer essentially as described by the manufacturer. The N-terminal sequence of the purified CART(55-102) was found to be:
  • mice were tested for appetite suppression as described in Example 9 after treatment with 0.1-1.0 micrograms of a test substance consisting of recombinant CART(55-102) dissolved in phosphate buffered saline. Intra-cerebroventricular injections of the test substance produced a statistically significant suppression of milk consumption.
  • the human CART(55-102) peptide corresponding to SEQ ID No. 5 differs from the rat form by having a valine residue in stead of a isoleucine residue in position 1. It may be prepared using the same method as described for the rat form starting from a human tissue or simply by substituting valine for isoleucine in position 1 of the rat form.
  • mice were tested for appetite suppression as described in Example 9 after treatment with 0.1- 2.0 ⁇ g of a test substance consisting of fragmented recombinant rat CART(55-102) (fragmented by trypsin and endopeptidase Asp-N) dissolved in phosphate buffered saline. Intra-cerebroventricular injections of the test substance did not produce statistically significant suppression of milk consumption at any dose tested (see table).
  • mice were tested for appetite suppression as described in Example 9 after treatment with 0.1- 2.0 ⁇ g of a test substance consisting of recombinant rat CART(55-102) with a disrupted secondary structure (reduced and pyridylated) dissolved in phosphate buffered saline. Intra- cerebroventricular injections of the test substance did not produce statistically significant suppression of milk consumption at any dose tested (see table).
  • PBS phosphate buffered saline
  • Consumption of a special test food (a mash made from a 2: 1 mixture of water and dry standard chow) was measured for one hour following the injection.
  • the degree of appetite suppression produced by a given test substance was determined by statistical comparison of the amount of food consumed by control rats (vehicle treated) with that of rats treated with a test substance.
  • the degree of feeding suppression in a group of rats was expressed as percent reduction of consumption relative to the mean amount consumed by the control group.
  • Example 21 Test for appetite suppression in rats bv recombinant rat CART(55-102. long).
  • SEO ID No. 4 Rats were tested for appetite suppression as described in Example 20 after treatment with 1 ⁇ g of a test substance consisting of recombinant rat CART(55-102) dissolved in phosphate buffered saline. Intra-cerebroventricular injection of 1 ⁇ g of the test substance produced a statistically significant 52% suppression of food consumption.
  • MOLECULE TYPE protein

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Child & Adolescent Psychology (AREA)
  • Obesity (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un polypeptide de produit de transcription régulé de cocaïne et d'amphétamine (CART) ou un de ses fragments ou une de ses variantes remplissant une fonction ou exerçant une activité de régulation de l'appétit, un produit de recombinaison d'acide nucléique codant ce polypeptide et un procédé servant à produire ce polypeptide. Elle concerne, de plus, des vecteurs de recombinaison comprenant ce produit de recombinaison d'acide nucléique codant le polypeptide, des cellules hôtes de recombinaison comprenant le produit de recombinaison d'acide nucléique ou le vecteur de recombinaison.
EP98909375A 1997-03-26 1998-03-26 Polypeptide exer ant une activite de regulation de l'appetit Withdrawn EP0971957A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DK35897 1997-03-26
DK35897 1997-03-26
DK131597 1997-11-19
DK131597 1997-11-19
PCT/DK1998/000128 WO1998042747A1 (fr) 1997-03-26 1998-03-26 Polypeptide exerçant une activite de regulation de l'appetit

Publications (1)

Publication Number Publication Date
EP0971957A1 true EP0971957A1 (fr) 2000-01-19

Family

ID=26063851

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98909375A Withdrawn EP0971957A1 (fr) 1997-03-26 1998-03-26 Polypeptide exer ant une activite de regulation de l'appetit

Country Status (4)

Country Link
EP (1) EP0971957A1 (fr)
JP (1) JP2002538757A (fr)
AU (1) AU6393498A (fr)
WO (1) WO1998042747A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EE9900497A (et) 1997-04-15 2000-06-15 Csir Söögiisu pärssiva toimega farmatseutilised kompositsioonid
GB2396815B (en) 1999-10-27 2004-09-08 Phytopharm Plc A composition comprising a pregnenone derivative and an NSAID
GB2363985B (en) 2000-06-30 2004-09-29 Phytopharm Plc Extracts,compounds & pharmaceutical compositions having anti-diabetic activity and their use
WO2002008275A2 (fr) * 2000-07-26 2002-01-31 Bayer Corporation Nouveaux genes exprimes dans l'hypothalamus des rats obeses
KR20100071984A (ko) * 2007-09-11 2010-06-29 몬도바이오테크 래보래토리즈 아게 치료제（들）로서의 ｌｖｖ―헤모르핀―６ 및 임의의 ａｆ１２１９８의 용도
AU2009226966A1 (en) * 2008-03-20 2009-09-24 Msd Oss B.V. Preparation of a pharmaceutical composition for increasing bone mineral density
CN112759634B (zh) * 2021-01-21 2022-04-08 浙江辉肽生命健康科技有限公司 具有氨基酸结构feyieenky的生物活性肽及其制备方法和应用
CN112724239B (zh) * 2021-01-22 2022-04-08 浙江辉肽生命健康科技有限公司 具有氨基酸结构nkeldpvqklfvdkireyk的生物活性肽及其应用
WO2023078478A1 (fr) * 2021-11-08 2023-05-11 Ustav Organicke Chemie A Biochemie Av Cr, V. V. I. Analogues lipidisés du peptide de transcription régulé par la cocaïne et l'amphétamine en tant qu'agents anti-obésité et neuroprotecteurs

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034619A1 (fr) * 1995-05-04 1996-11-07 Amgen Inc. Procedes de prevention de la degenerescence des neurones et de promotion de la regeneration de ceux-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9842747A1 *

Also Published As

Publication number Publication date
JP2002538757A (ja) 2002-11-12
WO1998042747A1 (fr) 1998-10-01
AU6393498A (en) 1998-10-20

Similar Documents

Publication Publication Date Title
US5912229A (en) Use of a pharmaceutical composition comprising an appetite-suppressing peptide
DE69717092T2 (de) Gebrauch einer pharmazeutischen Zusammensetzung, enthaltend ein appetitzügelndes Peptid
CN1483831B (zh) 获得具有正确键合的胱氨酸键的胰岛素或胰岛素衍生物的方法
WO1996022308A2 (fr) Facteur de suppression d'appetit et procedes connexes
KR100393508B1 (ko) 인간 인터루킨 4의 안타고니스트 또는 부분적 아고니스트로서의 신규hIL-4 변이단백질
WO1996022308A9 (fr) Facteur de suppression d'appetit et procedes connexes
CN1129955A (zh) 截短的生物活性更高的角质形成细胞生长因子
JPH03504597A (ja) インスリン様成長因子活性の強化および阻害方法
CN101243190A (zh) 制备成熟胰岛素多肽的方法
EP0871474A1 (fr) Production d'insuline humaine
WO2006018428A2 (fr) Proteines hybrides d'hemopexine
HU215434B (hu) Eljárás interleukin-1 inhibitorok, ezeket kódoló DNS-szekvenciák, vektorok és gazdasejtek előállítására
WO1998042747A1 (fr) Polypeptide exerçant une activite de regulation de l'appetit
DE69836075T2 (de) Verfahren zur spaltung von fusionproteinen
Pohlig et al. Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae
CZ54897A3 (en) Dimer of trefoil peptide
Adolf et al. Human interferon ω1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein
US6458927B1 (en) Polypeptide with appetite regulating activity
JPH06507632A (ja) 成長ホルモンアンタゴニスト
AU615194B2 (en) Bovine growth hormone analogs
Kannan et al. Human pS2/trefoil factor 1: production and characterization in Pichia pastoris
Nishizawa et al. Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae
Ishikawa et al. Production of human calcitonin in Escherichia coli from multimeric fusion protein
EP0416029A1 (fr) Peptides de procalcitonine
Churgay et al. Purification and characterization of secreted human leptin produced in baculovirus-infected insect cells

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19991026

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL PT SE

AX Request for extension of the european patent

Free format text: AL PAYMENT 19991026;LT PAYMENT 19991026;LV PAYMENT 19991026;MK PAYMENT 19991026;RO PAYMENT 19991026;SI PAYMENT 19991026

RIN1 Information on inventor provided before grant (corrected)

Inventor name: KRISTENSEN, PETER

Inventor name: JUDGE, MARTIN, EDWARD

Inventor name: THIM, LARS

Inventor name: CHRISTIANSEN, KENNET

Inventor name: HASTRUP, SVEN

17Q First examination report despatched

Effective date: 20031010

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040421