KR19980701798A - Obesity-inhibiting protein - Google Patents

Abstract

The present invention provides an obesity-suppressing protein that is administered to a patient to regulate adipose tissue. Such reagents thus allow patients to overcome their obesity deficiencies and to reduce their risk of type II diabetes, cardiovascular disease and cancer to a normal life.

Description

Obesity-inhibiting protein

The present invention belongs to the field of medicine for the treatment of diseases related to human medicine, especially obesity and obesity. More particularly, the present invention relates to an obesity-suppressing protein that regulates adipose tissue upon administration to a patient.

Obesity and especially upper body obesity are very serious public health problems that are common in the United States and globally. According to recent statistics, over 25% of the US population and over 27% of the Canadian population have exceeded their standard weight [Kuczmarski, Amer. J. of Clin. Nutr. 55: 495s-502s (1992); Reeder et. al., Can. Med. Ass. J. , 23: 226-233 (1992)). Abdominal obesity is the strongest risk factor for type II diabetes mellitus and is equally a powerful risk factor for cardiovascular disease and cancer. The estimated cost of health care for recent obesity is around $ 150 billion worldwide. As the problem became serious enough, the US Public Health Director began taking the lead in fighting the steadily growing excess of fat in American society.

Most of these obesity-induced pathologies are thought to be strongly associated with dyslipidemia, hypertension and insulin resistance. Many studies have shown that the reduction of obesity by diet and exercise significantly reduces these risk factors. Unfortunately, these treatments are not nearly as successful, with failure rates reaching 95%. These failures may be due to the fact that such conditions are strongly associated with an increased appetite, preference for high caloric food, reduced physical activity, and genetically inherited factors that contribute to increased lipid metabolism. This means that those who inherit these genetic traits tend to become obese, despite their efforts to defeat this condition. Thus, there is a need for a medicinal substance that will treat this hyperlipidemic disorder and allow the physician to treat obese patients despite genetic inheritance.

Physiologists have hypothesized that for many years, excess fat produced when mammals overeated signals to the brain that the body is obese, causing the body to eat less and consume more fuel [G. R. Hervey, Nature 227: 629-631 (1969)]. This feedback model is supported by parabiotic experiments, which means that circulating hormones regulate obesity.

The ob / ob mouse is a model of obesity and diabetes and is known to possess an autosomal recessive trait associated with mutations within chromosome 6. Recently Yiying Zhang and co-workers have published positional cloning of mouse genes related to these conditions [Yiying Zhang et al ., Nature 372 : 425-32 (1994)]. This report discloses a gene encoding a protein consisting of 167 amino acids with a signal peptide consisting of 21 amino acids exclusively expressed in adipose tissue. Similarly, the cloning and expression of the rat obesity gene (ob gene) is disclosed in Murakami et al., Biochemical and Biophysical Research Communications 209 (3): 944-52 (1995). In view, the protein encoded by the ob gene is currently estimated to be a lipid-modulating hormone. Zhang et al. Report no pharmacological activity.

However, the inventors have discovered that the protein disclosed by Zhang et al. Is a very poor drug substance due to chemical and / or physical instability. Human proteins tend to be more prone to precipitation, for example. Pharmaceutical agents of natural protein containing precipitates increase the risk of causing an immune response in the patient. Thus, there remains a need to develop medicinal substances useful for providing improved physical and chemical stability and helping patients to control their appetite and metabolism.

Most importantly, it has been found to the present invention that the specific replacement of 77, 97 to 111, 118 and / or 138 amino acid residues of human obesity protein is an excellent therapeutic agent with improved stability. Thus, the present invention provides a biologically active obesity protein. The protein of the present invention can be more easily formulated and stored. Moreover, the present compounds are pharmaceutically excellent and carry excellent therapeutic doses. Thus, these drugs help patients overcome their obesity disorders and normalize their risk for type II diabetes, cardiovascular disease, and cancer, so they can live a normal life.

SUMMARY OF THE INVENTION

The present invention relates to the protein of SEQ ID NO: 1 (Formula I) or a pharmaceutically acceptable salt thereof.

Wherein Xaa at position 4 is Gln or Glu,

Xaa at position 7 is Gln or Glu,

Xaa at position 22 is Asn, Asp, or Glu,

Xaa at position 27 is Thr or Ala,

Xaa at position 28 is Gln, Glu or absent,

Xaa at position 34 is Gln or Glu,

Xaa at position 54 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly,

Xaa at position 56 is Gln or GIu,

Xaa at position 62 is Gln or GIu,

Xaa at position 63 is Gln or GIu,

Xaa at position 68 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly,

Xaa at position 72 is Asn, Asp or Glu,

Xaa at position 75 is Gln or Glu,

Xaa at position 77 is Ser or Ala,

Xaa at position 78 is Gln, Asn or Asp,

Xaa at position 82 is Gln, Asn or Asp,

Xaa at position 118 is Gly or Leu,

Xaa at position 130 is Gln or Glu,

Xaa at position 134 is Gln or GIu,

Xaa at position 136 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly,

Xaa at position 139 is Gln or GIu,

The protein

His at position 97, which is substituted with Gln, Asn, Ala, Gly, Ser or Pro,

Trp at position 100 which is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu,

Ala at position 101 substituted with Ser, Asn, Gly, His, Pro, Thr or Val,

Ser at position 102 substituted with Arg,

Gly at position 103 substituted with Ala,

Glu at position 105 substituted with Gln,

Thr at position 106 substituted with Lys or Ser,

Leu at position 107 substituted with Pro,

Asp at position 108 substituted by Glu,

Gly at position 111 which is substituted with Asp,

Trp at position 138 substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu.

The present invention also provides a method of treating obesity comprising administering the protein of SEQ ID NO: 1 to a mammal in need thereof.

The present invention also provides a pharmaceutical composition comprising the protein of SEQ ID NO: 1 and at least one pharmaceutically acceptable diluent, carrier or excipient therefor.

A further aspect of the present invention is

(a) transforming a host cell with a DNA encoding a protein of SEQ ID NO: 1 having an arbitrary leader sequence;

(b) culturing the host cell and isolating the protein encoded in step (a); Optionally,

(c) cleaving the leader sequence with an enzyme to produce the protein of SEQ ID NO: 1.

For purposes of the present invention, the following terms and abbreviations used in this specification and claims are defined as follows.

Base pair (bp) - refers to DNA or RNA. The abbreviations A, C, G and T denote the 5'-monophosphate forms of nucleotide (deoxy) adenine, (deoxy) cytidine, (deoxy) guanine and Corresponding. The abbreviations U, C, G and T correspond to the 5'-monophosphate forms of the nucleosides uracil, cytidine, guanine and thymine, respectively, when they are in the RNA molecule. In double-stranded DNA, the base pair can refer to a combination of A and T or C and G. In a DNA / RNA heteroduplex, the base pair can refer to a combination of T and U or C and G.

DNA-deoxyribonucleic acid.

EDTA - Ethylenediaminetetraacetic acid.

Fragments of antibodies capable of binding to an antigen, having properties similar to the original antibody molecule from which they are derived, including immunoreactive protein (s) -antibodies, and those described in PCT Application No. PCT / US 87/02208, The term used collectively to describe short chain polypeptide binding molecules such as those described in WO 88/01649.

mRNA-messenger RNA.

Plasmid - a self-replicating gene component other than a chromosome.

PMSF - Abbreviation for phenylmethylsulfonyl fluoride

The reading frame - the triplet as a nucleotide sequence in which translation is read by a translator of tRNA, ribosomes and related factors into a triplet, corresponds to a particular amino acid. Since each triplet is distinguishable, its length is the same, the coding sequence must be a multiple of three. Once a base pair is inserted or removed (termed a structural displacement mutation), two different proteins can be generated that are encoded by the same DNA fragment. To prevent this, the triplet codon corresponding to the desired polypeptide has to be aligned in multiples of 3 from the initiation codon, i.e. the correct reading structure has to be maintained.

Recombinant DNA cloning vector - Any self-replicating agent, including, but not limited to, plasmids and phage comprising DNA molecules into which one or more additional DNA fragments can be inserted or inserted.

Recombinant DNA expression vector - All recombinant DNA cloning vectors into which a promoter has been introduced.

Replicon-plasmid or other vector.

RNA-ribonucleic acid

RP-HPLC - reversed phase high performance liquid chromatography.

The process by which the information contained in the transcription-DNA nucleotide sequence is transferred into the complementary RNA sequence.

Translation - The process by which the genetic information of messenger RNA is used to designate and direct the synthesis of polypeptide chains.

Tris - Tris (hydroxymethyl) aminomethane abbreviation.

Treating - treating and managing a patient for the purpose of defeating a disease, condition, or disorder; preventing the onset of symptoms or complications, alleviating symptoms or complications, or eliminating a disease, condition, or disorder; And administering the compounds of the present invention. Therefore, obesity treatment includes inhibition of food intake, suppression of weight gain, and induction of weight loss in patients in need thereof.

Vector - replicons used in the transformation of cells in genetic engineering, including polynucleotide sequences corresponding to appropriate protein molecules, which, when combined with appropriate regulatory sequences, confer specific properties on the transforming host cell. Plasmids, viruses and bacteriophages are suitable vectors because they are replicons themselves. Artificial vectors are made by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. The vector includes a recombinant DNA cloning vector and a recombinant DNA expression vector.

X-gal - Abbreviation for 5-bromo-4-chloro-3-indolylbeta-D-galactoside.

The amino acid abbreviation is accepted by the USPTO as described in 37 CFR § 1.822 (b) (2) (1993). Those skilled in the art will recognize that certain amino acids tend to rearrange. For example, Asn is described in [I. Schoen et al. , Int. J. Peptide Protein Res. 14 : 485-94 (1979)) and aspartic acid and isoaspartate as described in the references cited in this document. Such rearranged derivatives are also included within the scope of the present invention. Unless otherwise stated, the amino acid is in L configuration.

As described above, the present invention provides the protein of SEQ ID NO: 1. A preferred protein is the protein of SEQ ID NO: 2 (Formula II) or a pharmaceutically acceptable salt thereof.

Wherein Asn at position 22 is optionally Gln or Asp,

Thr at position 27 is optionally Ala,

Gln at position 28 is optionally Glu or absent,

Met at position 54 is optionally Ala,

Met at position 68 is optionally Leu,

Asn at position 72 is optionally Glu or Asp,

Ser at position 77 is optionally Ala,

Gly at position 118 is arbitrarily Leu

The protein

His at position 97, which is substituted with Gln, Asn, Ala, Gly, Ser or Pro,

Trp at position 100 which is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu,

Ala at position 101 substituted with Ser, Asn, Gly, His, Pro, Thr or Val,

Ser at position 102 substituted with Arg,

Gly at position 103 substituted with Ala,

Glu at position 105 substituted with Gln,

Thr at position 106 substituted with Lys or Ser,

Leu at position 107 substituted with Pro,

Asp at position 108 substituted by Glu,

Gly at position 111 substituted with Asp or

Trp at position 138 substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu.

A preferred protein is a protein of SEQ ID NO: 2 wherein Trp at position 100 is Gln, Tyr, Phe, Ile, Val or Leu, or Trp at position 138 is Gln, Tyr, Phe, Ile, Val or Leu.

Another preferred protein is the protein of SEQ ID NO: 3 (Formula III) or a pharmaceutically acceptable salt thereof.

(Wherein, His at position 97 is substituted with Gln, Asn, Ala, Gly, Ser or Pro,

Trp at position 100 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu,

Ala at position 101 is substituted with Ser, Asn, Gly, His, Pro, Thr or Val,

Ser at position 102 is substituted with Arg,

Gly at position 103 is substituted with Ala,

Glu at position 105 is substituted with Gln,

Thr at position 106 is substituted with Lys or Ser,

Leu at position 107 is substituted with Pro,

Asp at position 108 is substituted with Glu,

Gly at position 111 is substituted with Asp, or

Trp at position 138 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu.

The most preferred protein is

His at position 97 is substituted with Gln, Asn, Ala, Gly, Ser or Pro,

Trp at position 100 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu,

Ala at position 101 is substituted with Ser, Asn, Gly, His, Pro, Thr or Val,

Glu at position 105 is substituted with Gln,

Thr at position 106 is substituted with Lys or Ser,

Leu at position 107 is substituted with Pro,

Asp at position 108 is substituted with Glu,

Gly at position 111 is substituted with Asp, or

Wherein the Trp at position 138 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu.

A more preferred protein of SEQ ID NO: 3 is

His at position 97 is substituted with Ser or Pro,

Trp at position 100 is substituted with Ala, Gly, Gln, Val, Ile or Leu,

Ala at position 101 is substituted with Thr, or

Trp at position 138 is a protein substituted with Ala, Ile, Gly, Gln, Val or Leu.

An additional preferred protein of SEQ ID NO: 3 is

His at position 97 is substituted with Ser or Pro,

Trp at position 100 is substituted with Ala, Gln or Leu,

Ala at position 101 is substituted with Thr, or

Trp at position 138 is a protein substituted with Gln.

Further preferred proteins of the invention include proteins of SEQ ID NO: 3 where amino acid residues at positions 97, 100, 101, 105, 106, 107, 108 and 111 are substituted as in Table 1 below.

The most preferred species of SEQ ID NO: 3 and Table 1 includes the species of SEQ ID NOs: 4 to 11.

The present invention provides a biologically active protein that provides effective treatment for obesity. Unexpectedly, the claimed proteins have improved properties by specifically substituting human obesity proteins. The claimed proteins are more stable than the mouse and human obesity proteins and thus are excellent therapeutic agents.

The claimed proteins are typically prepared by recombinant techniques. Techniques for forming substitution mutations at predetermined positions in DNA containing already known sequences are well known, for example, as M13 primer mutagenesis. A mutation that may occur in the DNA encoding the obesity-suppressing protein of the invention should not occur in a sequence outside the reading structure, and preferably will not result in a complementary region capable of forming a secondary mRNA structure [DeBoer et al. European Patent No. 75,444A (1983)].

The compounds of the present invention can be prepared by recombinant DNA techniques or by semisynthetic methods in well known chemical procedures, for example, in solutions starting with liquid or solid phase peptide synthesis or protein fragments paired via conventional solution methods have.

A. Solid phase

The claimed protein can be synthesized by solid phase peptide synthesis or recombinant method. The principles of solid phase chemical synthesis of polypeptides are well known in the art and can be found in general textbooks such as Dugas, H. and Penney, C, Bioorganic Chemistry Springer-Verlag, New York, 54-92 (1981) . For example, peptides can be synthesized by solid-phase methods using synthetic cycles provided by PE-Applied Biosystems 433A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA) and Applied Biosystems, Inc. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical suppliers. For the preparation of the C-terminal carboxamide, sequential Boc chemistry using the double-coupled protocol is applied to the starting p-methylbenzhydrylamine resin for the preparation of the C-terminal carboxamide. The corresponding PAM resin is used for the preparation of the C-terminal amino acid. Arginine, asparagine, glutamine, histidine and methionine are paired using a pre-prepared hydroxybenzotriazole ester. The following side chain protection can be used.

Arg, tosyl

Asp, cyclohexyl or benzyl

Cys, 4-methylbenzyl

Glu, cyclohexyl

His, benzyloxymethyl

Lys, 2-chlorobenzyloxycarbonyl

Met, sulfoxide

Ser, benzyl

Thr, benzyl

Trp, formyl

Tyr, 4-bromocarbobenzoic acid.

Boc deprotection can be performed with trifluoroacetic acid (TFA) in methylene chloride. Removal of formyl from Trp is carried out by treating the peptide resin with 20% piperidine in dimethylformamide at 4 占 폚 for 60 minutes. Met (O) can be reduced by treating the peptidyl resin with TFA / dimethyl sulfide / concentrated HCl (95/5/1) at 25 ° C for 60 minutes. Following the pretreatment described above, the peptide is further deprotected and the peptide is eluted with 10% m-cresol or m-cresol / 10% p-thiocresol or m-cresol / p-thiocresol / Using anhydrous hydrogen fluoride, it is cut out from the resin. Cleavage of the side chain protecting group (s) and cleavage of the peptide from the resin is carried out at 0 ° C or less, preferably at -20 ° C for 30 minutes and then at 0 ° C for 30 minutes. After removing HF, the peptide / resin is washed with ether. The peptide is extracted with glacial acetic acid and lyophilized. The purification is carried out on a reverse phase C18 chromatography (Vydac) column in 0.1% TFA with a gradient of increasing acetonitrile concentration.

The skilled artisan recognizes that solid phase synthesis can also be performed using the FMOC method and the TFA / scavenger cleavage mixture.

B. recombinant synthesis

The claimed proteins can also be prepared by recombinant methods. Recombinant methods are preferred when high yields are desired. The basic steps in the recombinant production of proteins include the following steps:

a) preparing a synthetic or semisynthetic (or isolated from natural source) DNA encoding the claimed protein,

b) integrating the coding sequence into an expression vector in a manner suitable for expressing the protein, either alone or in the form of a fusion protein,

c) transforming a suitable eukaryotic or prokaryotic host with an expression vector, and

d) recovering and purifying the protein produced by recombination.

a. Gene production

Synthetic genes that are used to produce proteins via in vitro or in vivo transcription and translation can be prepared by techniques well known in the art. It will be appreciated by those skilled in the art that the genetic code is naturally degenerative and that it is capable of producing a suitably sized but deterministic number of DNA sequences encoding the claimed protein. In a preferred method of the invention, DNA synthesis is performed with recombinant DNA technology.

Methods for producing synthetic genes are well known in the art. See, for example, Brown et al., Methods in Enzymology , Academic Press, NY, vol. 68, 109-115 (1979). The DNA sequence corresponding to the claimed protein synthetase was prepared using a conventional DNA synthesizer such as the Applied Biosystems 380A type or 380B type DNA synthesizer (product of Applied Biosystems, Inc., Lincoln Center Drive 850, Foster City, CA 94404) can do.

In some applications it is desirable to insert a convenient protease cleavage site between the signal peptide and the structural protein to modify the coding sequence of the claimed protein in order to facilitate the resected cleavage of the signal peptide from the fusion protein structure Lt; / RTI >

The gene encoding the claimed protein may also be produced using polymerase chain reaction (PCR). The template may be a cDNA library (commercially available from CLONETECH or STRATAGENE) or an mRNA isolated from human adipose tissue. Such methods are well known in the art [Maniatis et al., Molecular Cloning : A Laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989)].

b. Direct expression or fusion protein

The claimed protein may be directly expressed or may be prepared by enzymatically or chemically cleaving the fusion protein in the form of a fusion protein containing the claimed protein. Various peptidases (for example, trypsin) such as diaminopeptidase that cleave a polypeptide at a specific position or digest a peptide from the amino terminal or carboxy terminal of the peptide chain are known. Moreover, certain chemicals (such as cyanogen bromide) will degrade polypeptide chains at specific locations. The skilled artisan will be aware of variations in the sequence necessary to insert a site-specific internal cleavage site into the amino acid sequence (and, if a recombinant method is used, a synthetic or semi-synthetic coding sequence). See, for example, Carter P., Site Specific Proteolysis of Fusion Proteins , Chapter 13 Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Society, Seed. (1990)].

c. Vector production

Standard binding techniques are used in the production of suitable vectors containing desired coding and control sequences. The isolated plasmid or DNA fragment is cut and recombined in the form necessary to cut and form the required plasmid.

To effect translation of the desired protein, a synthetic restriction endonuclease is used to insert the processed synthetic DNA sequence into a portion of a suitable amount of the recombinant DNA expression vector. Synthetic coding sequences are designed to have restriction endonuclease cleavage sites at the end of the transcript to facilitate their isolation and integration from the expression and amplification and expression plasmids. The isolated cDNA coding sequence is modified using a synthetic linker to facilitate insertion of this sequence into the desired cloning vector in a manner well known in the art. The particular endonuclease used will be specified by the restriction endonuclease cleavage form of the native expression vector to be used. The choice of restriction site is chosen to properly position the coding sequence with the regulatory sequence to make the expression of the claimed protein and the appropriate reading structure.

Typically, plasmid vectors containing promoter and regulatory sequences derived from host compatible species are used with these hosts. The vector typically carries a replication site as well as a marker sequence capable of providing phenotypic selection in the transformed cells. For example, E. E. coli is transformed using pBR322, a plasmid typically derived from E. coli species [Bolivar et al., Gene 2: 95 (1977)]. Because plasmid pBR322 contains genes with resistance to ampicillin and tetracycline, it provides an easy means to differentiate transformed cells. The pBR322 plasmid, or other microbial plasmid, should also be capable of containing or modifying the promoter and other regulatory elements commonly used in recombinant DNA technology.

The desired coding sequence is inserted at an appropriate position in the expression vector so that it is transcribed from the promoter and the ribosome binding site, and the promoter and ribosome binding site should be able to function in the host cell in which the protein is expressed. Examples of such expression vectors are the plasmids described in Belagaje et al., U. S. Patent No. 5,304, 493, the teachings of which are incorporated herein by reference. The gene encoding A-C-B proinsulin described in U.S. Patent No. 5,304,493 can be removed from the plasmid pRB182 using restriction enzymes NdeI and BamHI. The gene encoding the protein of the present invention can be inserted into the plasmid backbone on the NdeI / BamHI restriction fragment cassette.

d. Prokaryotic expression

Prokaryotes are typically used for the cloning of the DNA sequences necessary for the production of vectors useful in the present invention. For example, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains that may be used include E. coli B and E. coli X1776 (ATCC No. 31537). These examples, however, are not intended to be limited to these, but to illustrate them.

Prokaryotes are also used for expression. Such as E. coli W3110 [prototrophic ATCC No. 27325], bacilli such as Bacillus subtilis and intestinal tracts such as Salmonella typhimurium or Serratia marcescans The above-mentioned strain and various Pseudomonas species can be used as well as bacteria. Promoters suitable for use with prokaryotic hosts include, but are not limited to,? -Lactamase (vector pGX2907 [ATCC 39344] contains replicons and? -Lactamase genes) and the lactose promoter family [Chang et al., Nature, 275: 615 (1978) and Goeddel et al., Nature 281: 544 (1979)], alkaline phosphatase, tryptophan (trp) promoter system (vector pATH1 [ATCC 37695] is a trpE fusion protein regulated by the trp promoter, ) And a tac promoter (isolated from the plasmid pDR540 ATCC-37282). However, other functional bacterial promoters in which the nucleic acid sequence is generally known may be obtained by linking this promoter with DNA encoding the protein using a linker or adapter, which experts in the art would provide any necessary restriction sites It can be done. The promoter for use in the bacterial system will also contain a Shine-Dalgarno sequence operably linked to the DNA encoding the protein.

e. Eukaryotic expression

Proteins can be produced recombinantly in eukaryotic expression systems. Preferred promoters for regulating the electrons in mammalian host cells include various sources such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably cytomegalovirus , Or may be a promoter of a heterologous mammal, such as, for example, a beta-actin promoter. The early and late promoters of the SV40 virus can be readily obtained as an SV40 restriction fragment containing the cloning start point of the SV40 virus (Fier et al., Nature, 273: 113 (1978)). The entire SV40 gene can be obtained from the plasmid pBRSV, ATCC 45019. The immediate early promoter of the human cell expansion virus can be obtained from the plasmid pCMBβ (ATCC 77177). Of course, promoters obtained from host cells or related species can also be used herein.

Transcription of the DNA encoding the claimed protein by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. The amplifying agent is usually a cis-active element of about 10 to 300 bp of DNA, and acts on the promoter to increase transcription. The amplifying agent is a transcription unit 5 '[Laimins, L et al., PNAS 78: 993 (1981)] and 3' [Lusky, M. L. et al., Mol. Cell Bio. 3: 1108 (1983)], in the intron [Banerji, J. L. et al., Cell 33: 729 (1983)] as well as in the coding sequence itself [Osborne, T. F. et al., Mol. Cell Bio. 4: 1293 (1984)] and directions and positions are relatively independent. Many amplification sequences are currently known from mammalian genes [globin, RSV, SV40, EMC, elastase, albumin, a-fetoprotein and insulin]. Typically, however, amplifiers of eukaryotic cell viruses will be used. Examples include SV40 late amplification products, cell expansion virus initiation promoter amplification products, polyoma amplification products on the rear surface of replication origin, and adenovirus amplification products.

Expression vectors used in eukaryotic host cells (cells with nuclei obtained from yeast, fungi, insects, plants, humans or other multicellular organisms) will also contain sequences necessary for the termination of transcription to effect mRNA expression. This site is transcribed into the polyadenylation compartment in the untranslated region of the mRNA encoding the protein. The 3 ' non-translated region also includes an electron termination site.

The expression vector may comprise a selection gene, also referred to as a selectable marker. Examples of suitable selectable markers for mammalian cells include, but are not limited to, dihydrofolate reductase (DHFR, which may be derived from the Bq1II / HindIII restriction fragment of pJOD-10 [ATCC 68815]), thymidine kinase (simple herpesvirus thymidine kinase (Included on the BamHI fragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance gene (available from pNN414 yeast artificial chromosome vector [ATCC 37682]). When such selectable markers are successfully transferred to the mammalian host cell, the infected mammalian host cell can survive if placed under selective pressure. Two different categories of selection systems are widely used. The first category is the use of mutated cell lines that lose their ability to grow without the supplementary medium based on the metabolism of the cells. Two kinds of, for example, CHO DHFR ^- a cell (LM (TK-) ATCC CCL- 2.3) - cells (ATCC CRL-9096) and mouse LTK. These cells can not live without the addition of nutrients such as thymidine or hypoxanthine. These cells lack specific genes necessary for a complete nucleotide synthesis pathway, and can not survive unless the deficient nucleotide is provided as a supplemental medium. As an alternative to feeding the medium, the intact native DHFR or TK gene is inserted into the cells lacking the respective genes to change their growth requirements. Individual cells that are not transformed with DHFR or TK genes will not survive in non-supplemented media.

The second category does not require the use of a mutant cell line as a dominant selection related to the selection system used for a particular cell well. These systems typically use drugs that inhibit the growth of host cells. Such cells bearing the novel gene will survive the selection process by expressing a protein with drug resistance. Examples of such dominant selection include neomycin [Southern P and Berg, P., J. Molec. Appl. Genet. 1 : 327 (1982)], mycophenolic acid [Mulligan, RC and Berg, P. Science 209: 1422 (1980)], or hygromycin [Sugden, B. et al., Mol Cell. Biol. 5: 410-413 (1985). The three examples above each use bacterial genes with resistance to the appropriate drug G418 or neomycin, xgpt (mycophenolic acid) or hygromycin, each of which is under the control of eukaryotes.

The preferred vector for eukaryotic expression is pRc / CMV. pRc / CMV is marketed by Invitrogen Corporation of 3985 Boulevard, Sorrento Valley, San Diego, CA 92121 USA. To verify that the sequence in the plasmid produced is correct, the ligation mixture is used to transform E. coli K12 strain DH5a (ATCC 31446) and successful transformants are selected using appropriate antibiotic resistance. The plasmid was purified from the transformant and was transformed into E. coli by the method of Messing, et al., Nucleic Acids Res. 9: 309 (1981)].

The host cell can be transformed with the expression vector of the present invention to induce the promoter, and the transformant can be selected and cultured in a conventional nutrient medium modified to be suitable for amplifying the gene. Conditions such as temperature, pH, and the like will fit the conventionally used host cells selected for expression and experts will already recognize. Techniques for transforming cells with the above vectors are well known in the art (Maniatis et al., Molecular Cloning : A Laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) Current Protocols in Molecular Biology (1989)].

Suitable suitable host cells for expressing the vector encoding the claimed protein in higher eukaryotes include the African savannah monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL-1651), the transformed primary cell line Kidney cell line 293 [Gramham, FL et al., J. Gen Virol. BHK-21 (C-13), ATCC CCL-10, Virology 16: 147 (1962) ], Chinese hamster ovary cells ^{CHO-DHFR - (ATCC CRL-} 9096), mouse Sertoli (Sertoli) cells [TM4, ATCC CRL-1715, Biol. Reprod. ATCC CRL-1587), human cervical epithelial carcinoma cells (HeLa, ATCC CCL-2), canine kidney cells (MDCK, ATCC CCL-2) (BRL 3A, ATCC CRL-1442), human drainage lung cells (WI-38, ATCC HB-75), human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065) Cells (MMT 060562, ATCC CCL51).

f. Yeast expression

In addition to prokaryotes, eukaryotic microorganisms such as yeast media can also be used. Although many different strains are commercially available, eukaryotic microorganisms are the most widely used for Saccharomyces cerevisiae or common baker's yeast. For example, plasmid YRp7 [ATCC-40053, Stinchcomb et al ., Nature 282: 39 (1979), Kingsman et al., Gene 7: 141 (1979), Tschemper et al., Gene 10: 157 )] Is generally used. This plasmid contains the trp gene, which provides a selectable marker for a mutant strain that has failed to synthesize tryptophan and has lost its ability to grow {for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics 85: 12 )]}.

Suitable promoting sequences for use with yeast hosts include 3-phosphoglycerate kinase [found in plasmid pAP12BD ATCC 53231 and described in U.S. Patent 4,935,350 (June 19, 1990)] or enolase (Derived from plasmid pAC1 ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1 ATCC 57090, 57091), Zimomonas mobilis (U.S. Patent No. 5,000,000, dated March 19, 1991) A promoter for enzymes of other appropriate processes such as kinase, pyruvate carboxytechase, phosphofructokinase, glucoose-6-phosphate isomerase, phosphoglucose isomerase and glucokinase.

Other yeast promoters that are inducible promoters with the additional advantage that transcription is regulated by their growth conditions include alcohol dehydrogenase 2, isocytocrome C, acid phosphatase, nitrogen metabolism related degrading enzymes, metallothionein (included in plasmid vector pCL28XhoLHBPV ATCC 39475, U.S. Patent No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and promoters for enzymes involved in the utilization of maltose and galactose (GAL1 found in plasmid pRY121 ATCC 37658). Suitable vectors and promoters for use in yeast expression are further described in < RTI ID = 0.0 > Is disclosed in European Patent Publication No. 73,657A to R. Hitzeman et al. Yeast amplifiers (found with the CYC1 promoter on the plasmid YEpsec-hI1beta ATCC 67024), such as UAS Gal, obtained from Saccharomyces Isis Cervarix are also advantageously used as yeast promoters.

The following examples are provided to further illustrate the preparation of the claimed proteins. The scope of the present invention should not be construed as being made merely by the following embodiments.

Example 1

Vector manufacture

The gene of SEQ ID NO: 12 is assembled into about 220 bp and about 240 bp segments derived from chemically synthesized oligonucleotides.

The 220 bp segment consists of 7 overlapping oligonucleotides extending from the NdeI site to the XbaI site at position 220 in the coding region and ranging in length from 34 to 83 bases in length. The 240 bp segment consists of seven overlapping oligonucleotides extending from XbaI to the BamHI site and ranging in length from 57 to 92 bases in length.

To assemble these fragments, 7 oligonucleotides, respectively, are mixed at the same molar amount, usually at a concentration of about 1-2 picomoles / μl. Prior to assembly, most of the oligonucleotides at the 5 " -end of the oligonucleotide fragment are phosphorylated in standard kinase buffer with T4 DNA kinase using conditions specified by the supplier of the reactants. The mixture is heated to 95 < 0 > C and slowly cooled to room temperature over 1-2 hours to ensure that the annealing of the oligonucleotides occurs properly. The oligonucleotide is then ligated to each other and bound to a suitable cloning vector such as pUC18 or pUC19. Buffers and conditions are to be recommended by the supplier of the enzyme. The vector for the 220 bp fragment was truncated with NdeI and XbaI whereas the vector for the 240 bp fragment was truncated with XbaI and BamHI before use. The recombinant mixtures were used to transform E. coli DH10B cells (commercially available from Gibco / BRL) and the transformed cells were treated with tryptone-yeast (TY) plates containing 100 μg / ml of ampicillin, X-gal and IPTG and thinly coated on a plate. The overnight colonies were grown in liquid TY medium containing 100 μg / ml of ampicillin, and plasmids were isolated and DNA sequence analysis was performed. The plasmid with the correct sequence is kept in the assembly of the complete gene. This was carried out by gel-purification of the 220 bp and 240 bp fragments and combining these two fragments with an expression vector, such as pRB182, which was truncated with NdeI and BamHI prior to use to remove the coding sequence for A-C-B proinsulin.

Example 2

The plasmid containing the DNA sequence encoding the desired protein is cut into PmlI and Bsu36I. The recognition sequences for these enzymes are located within the coding region of the protein at the nucleotide at position 275 and at the nucleotide at position 360, respectively. The cloning vector does not include these recognition sequences. As a result, only two manipulations are observed after the digestion of the restriction enzymes PmlI and Bsu36I, one of which corresponds to a vector fragment and the other corresponds to an approximately 85 bp fragment that has disappeared within the protein coding sequence. This sequence can be replaced by any DNA sequence encoding the amino acid substitutions listed in Table 1. This DNA sequence is chemically synthesized as two oligonucleotides with terminal and complementary bases that are well terminated by digestion by PmlI and Bsu36I. The chemically synthesized oligonucleotides are mixed in the same molar amount (1 to 10 picomoles / 占 퐇), heated to 95 占 폚, and slowly lowered to 20 to 25 占 폚 to anneal. The annealed oligonucleotides are used in standard binding reactions. The bound product is transformed and analyzed as described in Example 1.

Example 3

The DNA sequence encoding the protein represented by the protein 255 having the Met Arg leader sequence in Table 1 was obtained using the plasmid and the sequence described in Example 2. The plasmid was cut into PmlI and Bsu36I and a synthetic DNA fragment of sequence 5 " -sequence 13 was annealed with sequence 5 ' -sequence 14

PmlI and Bsu36I sites. After binding, transformation and plasmid isolation, the sequence of the synthetic fragment was verified by DNA sequencing.

Example 4

The DNA sequence encoding sequence 4 with the Met Arg leader sequence was obtained using the sequence and plasmid described in Example 2. The plasmid was cut into PmlI and Bsu36I and a synthetic DNA fragment of sequence 5 " -sequence 15 was annealed with sequence 5 ' -sequence 16

PmlI and Bsu36I sites. After binding, transformation and plasmid isolation, the sequence of the synthetic fragment was verified by DNA sequencing.

Techniques for transforming cells with the above vectors are well known in the art (Maniatis et al., Molecular Cloning : A Laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, [ Current Protocols in Molecular Biology (1989)]. Techniques related to the transformation of E. coli cells used in the preferred practice of the invention exemplified herein are well known in the art. Suitable conditions under which the transformed E. coli cells are cultured depend on the nature of the E. coli host cell line and the nature of the expression vector or cloning vector used. For example, vectors incorporating the c1857 heat-induced lambda-phage promoter-operator site require temperature shifts of about 30 to about 40 DEG C in culture conditions to induce protein synthesis.

In a preferred embodiment of the present invention, E. coli K12 RV308 cells are used as host cells but many other cell lines such as E. coli K12, L201, L687, L693, L507, L640, L641, L695, L814 But are not limited to these. The transformed host cells are then thinly coated in a suitable medium at a selection pressure of an antibiotic corresponding to the resistance gene present in the expression plasmid. The cells were then incubated for a suitable temperature and time in the host cell system.

Proteins expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies containing large amounts of over-expressed proteins (Kreuger et al., Protein Folding , Gierasch and King, pp. 136-142 (1990), American Association for Advancement of Science Publication No. 89-18s, Washington, DC. Seed.]. Such proteins must be lysed to undergo further purification and isolation of the desired protein product. Id . Various techniques using a strongly denaturing solution such as guanidiphenyl-HCl and / or a weakly denaturing solution such as urea are used to dissolve the protein. Gradually remove the denaturant from the solution (often using dialysis) to allow the denatured protein to recover its original structure. The specific conditions for denaturation and folding are determined by the specific protein expression system and / or the protein of interest.

Example 5

The protein of Example 3 with the Met Arg leader sequence is expressed and isolated in E. coli, diluted with PBS or diluted with 8 M urea (all containing 5 mM cysteine), folded and thoroughly dialyzed with PBS. In this process, protein aggregation was scarcely observed. After final purification of the protein by size exclusion chromatography, the protein was concentrated to 3 to 3.5 mg / ml in PBS. In contrast to the natural human protein, which exhibits substantial aggregation during the concentration process, no aggregation of the protein was observed visually.

Human protein Ob was eluted at approximately 56.8% acetonitrile through reversed phase HPLC analysis, and the mouse protein eluted at 55.8% and the title protein with Met Arg leader sequence eluted at 53.7%. Thus, it has been unexpectedly found that human proteins with mouse inserts have higher hydrophilicity than human molecules or mouse molecules.

Example 6

The protein of SEQ ID NO: 4 with the Met Arg leader sequence was expressed in E. coli. The granules were separated and dissolved in 8 M urea containing 5 mM cysteine. The protein is purified by anion exchange chromatography and diluted with 8 M urea (5 mM cysteine), folded and thoroughly dialyzed with PBS by technique. The pH of the protein solution was lowered to about 2.8. Met Arg leader sequence was cut out by adding 6 to 10 milliliters of dDAP per mg of protein. The conversion reaction was carried out at room temperature for 2 to 8 hours. The progress of the reaction was observed by high performance reversed phase chromatography. The reaction was terminated by fitting the pH to 8 using NaOH. The des (Met-Arg) protein is further purified by cation exchange chromatography with 7 to 8 M of urea and purified by size exclusion chromatography with PBS. After final purification of the protein by size exclusion chromatography, the protein was concentrated to 3 to 3.5 mg / ml in PBS. Protein aggregation was not observed visually.

Preferably, the protein of the present invention is expressed together with the leader sequence. Although valid reader sequences are known to those of skill in the art, the preferred leader sequence is Met-R ₁ - (where R ₁ is any amino acid except Pro), so the expressed protein is readily available using Cathepsin C Can be converted to the claimed protein. Preferably, R ₁ is Arg, Asp or Tyr and most preferably a protein having the Met-Arg leader sequence is expressed. Interestingly, the leader sequence does not significantly affect the stability or activity of the active protein. However, the leader sequence is preferably truncated in the protein. Thus, the protein of sequence: Met-R ₁ - SEQ ID NO: ₁ is useful as a biological drug and is preferably useful as an intermediate.

Purification of the claimed proteins is carried out using techniques known in the art and includes reverse phase chromatography, affinity chromatography, ion exchange and size exclusion chromatography.

The claimed protein comprises two cysteine residues. Therefore, a disulfide bond is formed to stabilize the protein. The present invention includes not only the protein of SEQ ID NO: 1 or 2 but also the protein without such disulfide bond, wherein Cys at position 96 is bridged to Cys at position 146. The proteins of the present invention may exist as dimers, trimesters, tetramers and other multimers when specifically implicated. Such masses are also included within the scope of the present invention.

The present invention provides a method of treating obesity. The method comprises administering an effective amount of an obesity-suppressing protein to the organism in a dose of 1 to 1000 [mu] g / kg. A preferred dose is about 10 to 100 [mu] g / kg of active compound. A typical daily dose for an adult is 0.5 to 100 mg. In carrying out this method, the compound of SEQ ID NO: 1 may be administered in a once-a-day administration mode or by administering it several times a day. Therapeutic systems may require long-term administration. The administered dose or total obesity inhibition will be determined by the physician and will depend on such factors as the severity and nature of the disease, the patient's age and general health status, and the patient's tolerance to the compound.

The present invention further provides pharmaceutical preparations comprising the compounds of the present invention. The protein, preferably in the form of a pharmaceutically acceptable salt, can be prepared for parenteral administration for therapeutic or prophylactic treatment of obesity. For example, the compound of SEQ ID NO: 1 may be mixed with conventional pharmaceutical carriers and excipients. The composition comprising the claimed protein contains about 0.1 to 90% by weight of the active protein, preferably in soluble form, and more usually about 10 to 30% by weight. Moreover, the proteins of the present invention may be administered alone or in combination with drugs useful for the treatment of diabetes or other obesity-suppressing drugs.

For intravenous (iv) use, the protein is administered with the commonly used intravenous fluid (s) and administered by infusion fluid. Such fluids may be, for example, physiological saline, Ringer's solution or 5% dextrose solution.

For intramuscular pharmaceutical preparations, a sterilizing agent, preferably a protein of SEQ ID NO: 1 or 2, is dissolved in a suitable soluble salt form, for example a hydrochloride salt, to produce a pharmaceutical (e.g., aqueous) solution of pyrogen-free water (distilled water), physiological saline or 5% glucose solution It can be administered as a diluent. Compounds of the appropriate insoluble form can be prepared and administered as a suspension in an aqueous base or as an ester of a long chain fatty acid such as a pharmaceutically acceptable oil base, for example ethylene oleate.

The ability of the compounds of the present invention to treat obesity has been demonstrated in vivo as follows.

Biological test

Parabiotic studies show that the protein is secreted by peripheral adipose tissue and that the protein can regulate normal weight gain as well as submaxillary muscle. Therefore, most closely related biological tests are performed by injecting the test substance by various routes of administration (for example, by iv, sc, ip, or minipump or cannula) , Weight gain, plasma chemistry or hormones (glucose, insulin, ACTH, corticosterone, GH, T4).

Suitable test animals include normal mice (such as ICR) and obese mice (ob / ob, Avy / a, KK-Ay, tubby mousse, fat mousse). The ob / ob mouse model of obesity and diabetes is commonly used in the art to direct obesity disorders. Comparisons of non-specific effects for such injections were made in animals (db / db mice, fa / fa or cp / cp) that were considered to lack the receptor using vehicles with or without the same active drug composition in the same animal Rats) by observing the same variables or the active drug itself. Proteins that show activity in these models will exhibit similar activity in other mammals, especially humans.

Since a target tissue is expected to be a hypothalamus that regulates food intake and lipid production, a similar model can be used to directly target the test material to the brain (for example, icv injection through the lateral ventricle or third ventricle) (Eg, the nucleus, the atrioventricular nucleus, and the perirencular nuclei). The same variables as described above could be measured or the secretion of neurotransmitters known to regulate feeding or metabolism could be observed (eg, NPY, galantine, norepinephrine, dopamine, β-endorphin secretion).

Representative proteins outlined in Table 2 below were prepared according to the guidance and examples provided herein. The descriptions of proteins in Table 2 and Table 3 below refer to the substituted amino acids of SEQ ID NO: 3 as provided in SEQ ID NO: The amino acid sequences of the proteins in Tables 2 and 3 were confirmed by mass spectrometry and / or amino acid analysis. The ability of the protein of the present invention to treat obesity in ob / ob mice is also shown in Table 2.

A similar study was performed in perifusion or tissue bath systems using in vitro extracellular hypothalamic tissue. In this situation, secretion or electrophysiological changes of neurotransmitters are observed.

The physical and chemical properties of the compounds of the present invention are demonstrated as follows.

Shaking test

The starting solution was a purified ob protein in phosphate-buffered saline (PBS; Dulbecco's PBS, Gibco BRL, without calcium phosphate or magnesium phosphate, available from Life Technologies, Inc., Grand Island, NY). The protein concentration is typically determined by the absorbance at 280 nm. However, other methods are used for Ob proteins with a theoretical absorbance value of 0.5 or less at 280 nm for a 1 mg / ml solution in a 1 cm cuvette. The total integrated peak area is determined from 25 μl of sample injected into an analytical size exclusion chromatography (SEC) column (Superdex-75, Pharmacia), where the chromatography is run at ambient temperature in PBS and observed at 214 nm. This peak area is then compared with the total SEC peak area of the Ob protein for which the concentration was initially determined by the absorbance at 280 nm. From this assay, dilute with PBS to make the final concentration of each Ob protein approximately 1.6 mg / ml. An aliquot of this solution is adjusted to pH 5.0, pH 6.0, pH 7.0 and pH 8.0 using diluted acetic acid or dilute NaOH. This pH-adjusted solution is then quantified by UV absorption or SEC technology.

The Ob protein solution was then added to 15 ml Teflon balls (product of Curtin Matheson Scientific, Inc., Kentucky, USA) each having a diameter of 3.1 mm (1/8 inch), 2 ml glass autosampler bottle Varian Instrument Group, Sunnyvale, CA). Remove air bubbles from the solution in the vial while gently shaking. Fill the glass bottle to near the top and seal it with Teflon-coated rods and screw caps. Separate vials are each evaluated during the blanket test period.

Place the test vial in a rotating device in the incubator device at exactly 40 ° C. Rotate the vial from end to end at 30 revolutions per minute to allow the vial to move from top to bottom while the Teflon beads remain in solution.

After a predetermined time, the contents of the vial are removed and centrifuged at ambient temperature (Fisher Scientific Model 235C centrifuge). Protein concentrations in clear supernatants are determined again with UV absorbance or SEC technology. The percentage of Ob protein remaining in the solution is calculated as the Ob concentration in the pH-adjusted starting solution and the Ob protein concentration in the supernatant after the shaking test.

The chemical and physical stability of the compounds of the present invention are shown in Table 3. The protein description in Table 3 represents the substituted amino acid of SEQ ID NO: 3 provided in SEQ ID NO: 1. For example, Ala (100) represents the protein of SEQ ID NO: 3 substituted with Ala instead of Trp at position 100. For reference, human Ob protein and mouse Ob protein are also shown.

The compound is active in at least one of the above biological tests and is an obesity inhibitory drug. As such, they are useful in the treatment of obesity and obesity-induced back pain. However, protein is not only useful as a therapeutic drug, but as recognized by experts in the field, protein is also useful for the production of diagnostic antibodies and is also useful as an animal food additive for proteins. In addition, the compounds are useful for controlling weight in mammals for cosmetic purposes. Aesthetic purpose is to control body weight of mammals to improve body shape. Mammals are not necessarily obesity. Such an anxiety application forms part of the present invention.

The principles, preferred embodiments and manner of operation of the invention have been described in the foregoing specification. It should be understood, however, that the invention, which is to be protected herein, is not to be limited to the specific forms disclosed, since it is by way of example only and is not intended to be limited to the particular forms disclosed. Modifications and variations may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims (21)

The protein of SEQ ID NO: 1 (Formula I) or a pharmaceutically acceptable salt thereof. SEQ ID NO: 1 Wherein Xaa at position 4 is Gln or Glu, Xaa at position 7 is Gln or Glu, Xaa at position 22 is Asn, Asp, or Glu, Xaa at position 27 is Thr or Ala, Xaa at position 28 is Gln, Glu or absent, Xaa at position 34 is Gln or Glu, Xaa at position 54 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly, Xaa at position 56 is Gln or GIu, Xaa at position 62 is Gln or GIu, Xaa at position 63 is Gln or GIu, Xaa at position 68 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly, Xaa at position 72 is Asn, Asp or Glu, Xaa at position 75 is Gln or Glu, Xaa at position 77 is Ser or Ala, Xaa at position 78 is Gln, Asn or Asp, Xaa at position 82 is Gln, Asn or Asp, Xaa at position 118 is Gly or Leu, Xaa at position 130 is Gln or Glu, Xaa at position 134 is Gln or GIu, Xaa at position 136 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly, Xaa at position 139 is Gln or GIu, The protein His at position 97, which is substituted with Gln, Asn, Ala, Gly, Ser or Pro, Trp at position 100 which is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu, Ala at position 101 substituted with Ser, Asn, Gly, His, Pro, Thr or Val, Ser at position 102 substituted with Arg, Gly at position 103 substituted with Ala, Glu at position 105 substituted with Gln, Thr at position 106 substituted with Lys or Ser, Leu at position 107 substituted with Pro, Asp at position 108 substituted by Glu, Gly at position 111 which is substituted with Asp, Trp at position 138 substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu. The method of claim 1, wherein His at position 97, which is substituted with Gln, Asn, Ala, Gly, Ser or Pro, Trp at position 100 which is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Ala at position 101 substituted with Ser, Asn, Gly, His, Pro, Thr or Val, Ser at position 102 substituted with Arg, Gly at position 103 substituted with Ala, Glu at position 105 substituted with Gln, Thr at position 106 substituted with Lys or Ser, Leu at position 107 substituted with Pro, Asp at position 108 substituted by Glu, A Gly at position 111, which is substituted with Asp, or a pharmaceutically acceptable salt thereof. A protein of SEQ ID NO: 2 (Formula II) or a pharmaceutically acceptable salt thereof. SEQ ID NO: 2 Wherein Asn at position 22 is optionally Gln or Asp, Thr at position 27 is optionally Ala, Gln at position 28 is optionally Glu or absent, Met at position 54 is optionally Ala, Met at position 68 is optionally Leu, Asn at position 72 is optionally Glu or Asp, Ser at position 77 is optionally Ala, Gly at position 118 is optionally Leu, The protein His at position 97, which is substituted with Gln, Asn, Ala, Gly, Ser or Pro, Trp at position 100 which is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu, Ala at position 101 substituted with Ser, Asn, Gly, His, Pro, Thr or Val, Ser at position 102 substituted with Arg, Gly at position 103 substituted with Ala, Glu at position 105 substituted with Gln, Thr at position 106 substituted with Lys or Ser, Leu at position 107 substituted with Pro, Asp at position 108 substituted by Glu, Gly at position 111 substituted with Asp or Trp at position 138 substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu. The protein according to claim 3, wherein Trp at position 100 is Gln, Tyr, Phe, Ile, Val or Leu, or Trp at position 138 is Gln, Tyr, Phe, Ile, Val or Leu. A protein of SEQ ID NO: 3 (Formula III) or a pharmaceutically acceptable salt thereof. SEQ ID NO: 3 In the formula, His at position 97 is substituted with Gln, Asn, Ala, Gly, Ser or Pro, Trp at position 100 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu, Ala at position 101 is substituted with Ser, Asn, Gly, His, Pro, Thr or Val, Ser at position 102 is substituted with Arg, Gly at position 103 is substituted with Ala, Glu at position 105 is substituted with Gln, Thr at position 106 is substituted with Lys or Ser, Leu at position 107 is substituted with Pro, Asp at position 108 is substituted with Glu, Gly at position 111 is substituted with Asp, or Trp at position 138 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu. The method according to claim 5, wherein His at position 97 is substituted with Gln, Asn, Ala, Gly, Ser or Pro, Trp at position 100 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu, Ala at position 101 is substituted with Ser, Asn, Gly, His, Pro, Thr or Val, Glu at position 105 is substituted with Gln, Thr at position 106 is substituted with Lys or Ser, Leu at position 107 is substituted with Pro, Asp at position 108 is substituted with Glu, Gly at position 111 is substituted with Asp, or Wherein the Trp at position 138 is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu. The method according to claim 6, wherein His at position 97 is substituted with Ser or Pro, Trp at position 100 is substituted with Ala, Gly, Gln, Val, Ile or Leu, Ala at position 101 is substituted with Thr, Protein in which Trp at position 138 is substituted with Ala, Ile, Gly, Gln, Val or Leu. The protein according to any one of claims 1 to 7, wherein Cys at position 96 is disulfide bonded to Cys at position 146. The Cys at position 96 is disulfide bonded to the Cys at position 146, or a salt thereof. SEQ ID NO: 4 The Cys at position 96 is disulfide bonded to the Cys at position 146, and a salt thereof. SEQ ID NO: 5 The Cys at position 96 is disulfide bonded to the Cys at position 146, and a pharmaceutically acceptable salt thereof. SEQ ID NO: 6 The Cys at position 96 is disulfide bonded to the Cys at position 146, or a salt thereof. SEQ ID NO: 7 The Cys at position 96 is disulfide bonded to the Cys at position 146, or a salt thereof. SEQ ID NO: 8 A protein of the formula: Wherein R ¹ is any amino acid except Pro, Xaa at position 4 is Gln or Glu, Xaa at position 7 is Gln or Glu, Xaa at position 22 is Asn, Asp, or Glu, Xaa at position 27 is Thr or Ala, Xaa at position 28 is Gln, Glu or absent, Xaa at position 34 is Gln or Glu, Xaa at position 54 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly, Xaa at position 56 is Gln or GIu, Xaa at position 62 is Gln or GIu, Xaa at position 63 is Gln or GIu, Xaa at position 68 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly, Xaa at position 72 is Asn, Asp or Glu, Xaa at position 75 is Gln or Glu, Xaa at position 77 is Ser or Ala, Xaa at position 78 is Gln, Asn or Asp, Xaa at position 82 is Gln, Asn or Asp, Xaa at position 118 is Gly or Leu, Xaa at position 130 is Gln or Glu, Xaa at position 134 is Gln or GIu, Xaa at position 136 is Met, methionine sulfoxide, Leu, Ile, Val, Ala or Gly, Xaa at position 139 is Gln or GIu, The protein His at position 97, which is substituted with Gln, Asn, Ala, Gly, Ser or Pro, Trp at position 100 which is substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu, Ala at position 101 substituted with Ser, Asn, Gly, His, Pro, Thr or Val, Ser at position 102 substituted with Arg, Gly at position 103 substituted with Ala, Glu at position 105 substituted with Gln, Thr at position 106 substituted with Lys or Ser, Leu at position 107 substituted with Pro, Asp at position 108 substituted by Glu, Gly at position 111 which is substituted with Asp, Trp at position 138 substituted with Ala, Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu. 15. The protein of claim 14 wherein R < ¹ > is Arg. (a) transforming a host cell with a DNA encoding a protein claimed in any one of claims 1 to 13 having an arbitrary leader sequence, (b) culturing the host cell, isolating the protein encoded in step (a), and optionally, (c) preparing the claimed protein according to any one of claims 1 to 13 by cutting the leader sequence with an enzyme. 14. A method for producing a protein as claimed in any one of claims 1 to 13. The method of claim 16, wherein the leader sequence is Met-R ₁ -. 18. The method of claim 17, wherein the leader sequence is Met-Arg-. A pharmaceutical formulation comprising a protein claimed in any one of claims 1 to 13 and a pharmaceutically acceptable diluent, carrier or excipient therefor. A method for treating obesity comprising administering a protein as claimed in any one of claims 1 to 13 to a mammal in need thereof. The protein of any one of claims 1 to 13 for use as a medicament.