OBESITY PROTEIN INTERMEDIATES AND THEIR PREPARATION AND USE

Abstract: The present invention relates to anti-obesity proteins, which when administered to a patient regulate fat tissue. The invention provides the penultimate intermediate in the renaturation pathway for the obesity protein. The invention further provides the preparation of the intermediate and its use to prepare a biologically active obesity protein or analog thereof.

Description: PL- Obesity Protein Intermediates and Their Preparation and Use The present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. Most specifically the invention relates to an intermediate used to prepare obesity proteins that when administered to a patient regulate fat tissue. The production of proteins in heterologous host organisms leads to the formation of inactive, sparingly- soluble protein aggregates, i.e., inclusion bodies. The formation of such inclusion bodies is a result of the high protein concentrations in the cell arising from expression. In order to obtain biologically-active proteins, the inclusion bodies must be dissolved by denaturation and reduced, and then the three-dimensional structure of the protein in its native spatial form must be formed by the adjustment of suitable conditions. R. Jaenic e, Prog. Biophys. Molec. Biol. 49:117-237 (1987) . However, renaturation into the biologically active conformation is not a quantitative process -- a multitude of non-functional species and conformations of the protein may be formed. In order to shift the equilibrium to form the biologically active conformation, conditions are selected which, both prevent the establishment of improper protein conformation and do not hinder renaturation. Because it is difficult, if not impossible, to predict how a polypeptide chain renatures into a highly, ordered conformation, the conditions that favor the formation of the biologically active protein cannot be predicted. Recently, Yiying Zhang and co-workers published the positional cloning of the mouse gene linked with obesity and diabetes. Yiying Zhang et al. Nature 372: 425-32 (1994) . This report disclosed a gene coding for a 167 amino acid protein with a putative 21 amino acid signal peptide that is exclusively expressed in adipose tissue. This peptide is speculated to be an adiposity regulating hormone. Likewise, Murakami et al. , in Biochem. and Biophys. Res. Comm. 209(3) : 944-52 (1995) disclose the obese rat gene and protein. The present invention provides the penultimate intermediate in the renaturation pathway for the obesity protein. The invention further provides the preparation of the intermediate and its use to prepare a biologically active obesity protein or analog thereof. Most significantly, by passing through this intermediate, a substantial increase in yield of the biologically active protein is observed. The intermediate also provides significant process advantages that allow the large scale manufacture of an obesity protein. The present invention provides a properly folded intermediate of an obesity protein or analog thereof. More particularly, the present invention is directed to a properly folded protein of the Formula (I) : 146 (I) wherein: A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof; B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein; Rl and R2 are independently H or in conjunction with the sulfur to which it is bound forms a mixed disulfide; provided that both Ri and R2 are not H. The invention also provides a process of preparing a protein of Formula I, which comprises: mixing an obesity protein or analog thereof with a solution comprising a denaturant and a thiol reducing reagent at a concentration of about 1 to 100 mM at a pH from about 7 to about 12. The invention further provides a process of preparing a properly folded protein of the Formula (II) : 146 (II) wherein: A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof; B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein; which comprises: (a) Solubilizing obesity protein inclusion bodies in a solution comprising: a denaturant at a concentration sufficient to solubilize the protein, and a thiol reducing reagent at a concentration of about 1 to 100 mM, at a pH from about 7 to about 12; (b) Reducing the thiol and denaturant concentration of the solution to effect disulfide bond formation. Description of the Figures Figure 1 provides an HPLC chromatogram run on a Zorbax C-8 column with buffer A being 50 mM ammonium phosphate, 0.5% SDS, and 5 % n-propanol at pH 7.6 and buffer B being 50 mM ammonium phosphate, 0.5% SDS, and 50 % n-propanol at pH 7.6, The peak at about 385 seconds represents the biologically active obesity protein and the later eluting peak at about 573 seconds represent the claimed intermediate prepared in the examples. For purposes of the present invention, as disclosed and claimed herein, the following terms and abbreviations are defined as follows: Base pair (bp) — refers to DNA or RNA. The abbreviations A,C,G, and T correspond to the 5'- monophosphate forms of the nucleotides (deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy) thymine, respectively, when they occur in DNA molecules . The abbreviations U,C,G, and T correspond to the 5'- monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA heteroduplex, base pair may refer to a partnership of T with U or C with G. Denaturing reagent or denaturant -- is known to one skilled in the art. Examples of denaturing reagents are described in R. Jaenicke, Proσ. Bior>hvs. Molec. Biol. 49: 117-237 (1987) . Preferred reagents include urea, thiocyanate, and guanidine, most preferably, 6 to 8 M urea. Mixed disulfide -- refers to a group derived from a thiol reducing reagent capable of disulfide exchange between the reagent and the cysteinyl residue of the polypeptide. Obesity protein -- refers to the protein produced from the obesity gene following transcription and deletions of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g., from the N-terminal valine-proline to the C-terminal cysteine of the mature protein. The mouse obesity protein and human obesity protein are published in Zhang et al . Nature 372: 425-32 (1994). The rat obesity protein is published in Murakami et al. , Biochemical and Biophysical Research Comm. 209(3): 944-52 (1995). The numbering of amino acids in the present specification is consecutively from the amino terminus of the full length, mature protein. Such uniform numbering of amino acid residues is well accepted by those of ordinary skill in the art. In the human, urine, porcine, bovine, and rat obesity proteins, the cysteines associated with disulfide formation are at residues 96 and 146. The phrase "A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein" is intended to include the amino acids from the N-terminus to the first cysteine associated with di¬ sulfide bond formation. The phrase "B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the obesity protein" is intended to include the amino acids from the first cysteine to the C-terminal cysteine of the obesity protein. It is understood that the deletion of one or more amino acids in a natural variant or fragment as well as any amino acid additions, including additions to the Cys at position 146, that do not effect the novel and basic characteristics of the invention are included in the definitions of A and B. Renumbering the amino acid residues of the protein is unnecessary and may result in confusion. For example, particularly with the murine and human obesity protein, a desGln(28) variant has been observed. Such variant is intended to be included in the present invention. Obesity protein analog refers to an obesity protein having one or more amino acid substitutions, preferably less than five and most preferably less than three substitutions, and includes proteins disclosed of the Formula (III) : (SEQ ID NO: 1) 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg lie Asn Asp lie Ser His Thr Xaa Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly Cys (III) wherein: Xaa at position 28 is Gin or absent; said protein having at least one of the following substitutions: Gin at position 4 is replaced with Glu; Gin at position 7 is replaced with Glu; Asn at position 22 is replaced with Gin or Asp; Thr at position 27 is replaced with Ala; Xaa at position 28 is replaced with Glu; Gin at position 34 is replaced with Glu; Met at position 54 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Gin at position 56 is replaced with Glu; Gin at position 62 is replaced with Glu; Gin at position 63 is replaced with Glu; Met at position 68 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Asn at position 72 is replaced with Gin, Glu, or Asp; Gin at position 75 is replaced with Glu; Ser at position 77 is replaced with Ala; Asn at position 78 is replaced with Gin or Asp; Asn at position 82 is replaced with Gin or Asp; His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro; Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; Ser at position 102 is replaced with Arg; Gly at position 103 is replaced with Ala; Glu at position 105 is replaced with Gin; Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; Gly at position 111 is replaced with Asp; Gly at position 118 is replaced with Leu; Gin at position 130 is replaced with Glu; Gin at position 134 is replaced with Glu; Met at position 136 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu-; or Gin at position 139 is replaced with Glu; or a pharmaceutically acceptable salt thereof. A properly folded obesity protein or analog thereof is the protein in the conformation or tertiary structure resulting in a biologically active protein useful in treating obesity and those conditions associated with obesity such as diabetes, cardiovascular disease and cancer. Ob gene — refers to any nucleic acid sequence that hybridizes, and is at least 50 % homologous, preferably 70 % homologous, and most preferably 80 % homologous to the native ob gene sequences disclosed by Zhang et al . Nature 372: 425-32 (1994) and Murakami et al. , Biochemical and Biophysical Research Comm. 209(3) : 944-52 (1995). The ob gene product is expressed specifically in adipose tissue and regulates energy balance. Obesity protein inclusion bodies — refers to insoluble protein aggregates or cytoplasmic aggregates containing, at least in part, the obesity protein to be recovered. Similarly, inclusion bodies when preparing an obesity protein analog refers to insoluble protein aggregates or cytoplasmic aggregates containing, at least in part, the obesity protein analog to be recovered. Plasmid -- an extrachromosomal self-replicating genetic element. Reading frame — the nucleotide sequence from which translation occurs "read" in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment. To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame" must be maintained. Recombinant DNA Cloning Vector — any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added. Recombinant DNA Expression Vector — any recombinant DNA cloning vector in which a promoter has been incorporated. Replicon -- A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector. Rl and R2 a:re independently H or in conjunction with the sulfur to which it is bound forms a mixed disulfide; a mixed disulfide is recognized and understood to be derived from a thiol reducing reagent capable of disulfide exchange. Suitable thiol reagents include one or more mercapto reagents containing a free -SH that is capable of forming a mixed disulfide and operable in disulfide exchange. Thiol therefore includes, but is not limited to, cysteine, 2-mercaptoethanol, glutathionine, cysteamine, S- mercaptoethanol (BME) , and the like. Thiol reagents such as dithiothreitol (DTT) , dithioerythritol (DTE) are also included in the present invention; but are less preferred due to instability of the mixed disulfide formed with such reagents. Accordingly, preferred moieties represented by Ri or R2 include SO3, SCH2CH(NH2) (COOH) , SCH2CHNH2, SCH2CH2OH, and H2NCH(COOH)CH2CH2CONHCH(CH2S)CONHCH2COO _ . Provided, however, both Ri and R2 are not H. Transcription -- the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence. Translation -- the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain. Tris -- an abbreviation for tris(hydroxymethyl)- aminomethane. Treating -- describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating obesity therefore includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof. Vector — a replicon used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors. The amino acids abbreviations are accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. § 1.822 (b)(2) (1993). One skilled in the art would recognize that certain amino acids are prone to rearrangement. For example, Asp may rearrange to aspartimide and isoasparigine as described in I. Schδn et al., Int. J. Peptide Protein Res. 14: 485-94 (1979) and references cited therein. These rearrangement derivatives are included within the scope of the present invention. Unless otherwise indicated the amino acids are in the L configuration. As noted, the present invention provides a protein of the Formula (I) : 146 (i : wherein: A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof; B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein; Rl and R2 are independently H or in conjunction with the sulfur to which it is bound forms a mixed disulfide; provided that both Ri and R2 are not H . In a preferred embodiment , A is a polypeptide of the formula: (SEQ ID NO : 2 ) 5 10 15 R 3 - Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser 35 40 45 Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro 50 55 60 He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 65 70 75 He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp 80 85 90 95 Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser wherei : 3 is absent, Met, Met-R4, or a leader sequence; R4 is any amino acid except Pro; Xaa at position 28 is Gin or absent; Gin at position 4 is optionally replaced with Glu; Gin at position 7 is optionally replaced with Glu; Asn at position 22 is optionally replaced with Gin or Asp; Thr at position 27 is optionally replaced with Ala; Xaa at position 28 is optionally replaced with Glu; Gin at position 34 is optionally replaced with Glu; Met at position 54 is optionally replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Gin at position 56 is optionally replaced with Glu; Gin at position 62 is optionally replaced with Glu; Gin at position 63 is optionally replaced with Glu; Met at position 68 is optionally replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Asn at position 72 is optionally replaced with Gin, Glu, or Asp; Gin at position 75 is optionally replaced with Glu; Ser at position 77 is optionally replaced with Ala; Asn at position 78 is optionally replaced with Gin or Asp; or Asn at position 82 is optionally replaced with Gin or Asp. Other preferred embodiments include those wherein A is a polypeptide of the formula: (SEQ ID NO: 2) 5 10 15 R3 - Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser 35 40 45 Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro 50 55 60 He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 65 70 75 He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp 80 85 90 95 Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser ; wherein: Xaa at position 28 is Gin or absent. Yet additional preferred embodiments include proteins wherein B is a polypeptide of the Formula: (SEQ ID NO: 3) 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly ; wherein: His at position 97 is optionally replaced with Gin, Asn, Ala, Gly, Ser, or Pro; Trp at position 100 is optionally replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; Ala at position 101 is optionally replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; Ser at position 102 is optionally replaced with Arg; Gly at position 103 is optionally replaced with Ala; Glu at position 105 is optionally replaced with Gin; Thr at position 106 is optionally replaced with Lys or Ser; Leu at position 107 is optionally replaced with Pro; Asp at position 108 is optionally replaced with Glu; Gly at position 111 is optionally replaced with Asp; Gly at position 118 is optionally replaced with Leu; Gin at position 130 is optionally replaced with Glu; Gin at position 134 is optionally replaced with Glu; Met at position 136 is optionally replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Trp at position 138 is optionally replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or Gin at position 139 is optionally replaced with Glu. Other preferred embodiments include proteins wherein B is a polypeptide of the formula: (SEQ ID NO:3) 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly ; wherei : Trp at position 100 is optionally replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu. One skilled in the art would recognize that in the production of an obesity protein or analog thereof in a procaryote expression system, it is necessary to express the protein with a N-terminal extension appendaged to the N- terminus of the protein. The N-terminal extension is optionally cleaved from the protein prior to administration. Preferred N-terminal extensions are Met or Met-R4- wherein R is any amino acid except Pro. The present invention also includes proteins wherein A optionally includes one or more amino acid leader sequence that may be used for purification or other purposes. Most preferred leader sequences include Gly-Ser- His-Met (SEQ ID NO: 4), Met-Gly-Ser-Ser-His-His-His-His-His- His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met (SEQ ID NO: 5) , Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO: 6), Glu-Ala- Glu-Ala (SEQ ID NO: 7), Leu-Glu-Lys-Arg- (SEQ ID NO: 8), Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val- Pro-Arg-Gly-Ser-Pro (SEQ ID NO: 9), and Gly-Ser-Pro- (SEQ ID NO: 10) . Such an N-terminal extension and/or leader sequence does not effect the basic and novel characteristics of this invention. The compounds of Formula (I) are folding intermediates of the biologically active obesity protein. Preferred obesity proteins are the native sequences and analogs such as those described in Basinski et al., in U.S. applications serial number 08/383,638, filed February 6, 1995, and serial number 08/588,061, filed January 19, 1996, of the Formula (III) . The murine and bovine sequences are described in Hansen M. Hsiung and Dennis P. Smith, in U.S. application serial number 08/445,305, filed May 19, 1995 herein incorporated by reference. Most preferred proteins of the present invention include proteins of Formula (III) , SEQ ID SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14. SEQ ID NO:11 1 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Gin Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Gin Gin Leu Asp Leu Ser Pro 145 Gly Cys wherein: Xaa at position 28 is Gin or absent. SEQ ID NO:12 1 5 10 15 Val Pro He Trp Arg Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Ser Asp He Ser His Met Gin Ser Val Ser Ser 35 40 45 Lys Gin Arg Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Val 50 55 60 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala He Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Leu Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys 100 105 110 Pro Leu Pro Gin Ala Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ala Leu Gin Asp Met Leu Arg Gin Leu Asp Leu Ser Pro 145 Gly Cys SEQ ID NO:13 1 5 10 15 Val Pro He Cys Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 35 40 45 Lys Gin Arg Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Leu 50 55 60 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala He Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys 100 105 110 Pro Leu Pro Gin Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val 115 120 125 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Arg Gin Leu Asp Leu Ser Pro 145 Gly Cys wherein Xaa at position 28 is Gin or absent. Human Obesity Protein SEQ ID NO:14 1 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly Cys wherein: Xaa at position 28 is Gin or absent. Other preferred proteins of the present invention are those of Formula (III) , wherein: Gin at position 4 is replaced with Glu; Gin at position 7 is replaced with Glu; Asn at position 22 is replaced with Gin or Asp; Thr at position 27 is replaced with Ala; Gin at position 28 is replaced with Glu; Gin at position 34 is replaced with Glu; Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala; Gln at position 56 is replaced with Glu; Gin at position 62 is replaced with Glu; Gin at position 63 is replaced with Glu; Met at position 68 is replaced with methionine sulfoxide, or Leu; Asn at position 72 is replaced with Gin or Asp; Gin at position 75 is replaced with Glu; Asn at position 78 is replaced with Gin or Asp; Asn at position 82 is replaced with Gin or Asp; Gin at position 130 is replaced with Glu; Gin at position 134 is replaced with Glu; Met at position 136 is replaced with methionine sulfoxide, Leu, lie; or Gin at position 139 is replaced with Glu. Other preferred proteins are those of Formula (III) wherein: Asn at position 22 is replaced with Gin or Asp; Thr at position 27 is replaced with Ala; Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala; Met at position 68 is replaced with methionine sulfoxide, or Leu; Asn at position 72 is replaced with Gin or Asp; Asn at position 78 is replaced with Gin or Asp; Asn at position 82 is replaced with Gin or Asp; or Met at position 136 is replaced with methionine sulfoxide, Leu, or lie. Still yet additional preferred proteins are those of Formula (III) , wherein: Asn at position 22 is replaced with Gin or Asp; Thr at position 27 is replaced with Ala; Met at position 54 is replaced with Leu, or Ala; Met at position 68 is replaced with Leu; Asn at position 72 is replaced with Gin or Asp; Asn at position 78 is replaced with Gin or Asp; Asn at position 82 is replaced with Gin or Asp; or Met at position 136 is replaced with Leu, or lie. Most significantly, other preferred embodiments are specific substitutions to amino acid residues 97 to 111 and/or 138 of the proteins of SEQ ID NO: 1. These substitutions result in significantly improved stability and are superior therapeutic agents. These specific proteins are more readily formulated and are more pharmaceutically elegant, which results in superior delivery of therapeutic doses. Accordingly, preferred embodiments are proteins of the Formula (IV) : (SEQ ID N0:1) 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly Cys (IV) wherein: Xaa at position 28 is Gin or absent; said protein having at least one substitution selected from the group consisting of: His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro; Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; Ser at position 102 is replaced with Arg; Gly at position 103 is replaced with Ala; Glu at position 105 is replaced with Gin; Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; Gly at position 111 is replaced with Asp; or Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or a pharmaceutically acceptable salt thereof. Preferred proteins are proteins of the Formula (V) : (SEQ ID NO:15) 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly Cys (V) said protein having at least one substitution selected from the group consisting of: His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro; Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; Ser at position 102 is replaced with Arg; Gly at position 103 is replaced with Ala; Glu at position 105 is replaced with Gin; Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; Gly at position 111 is replaced with Asp; or Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or a pharmaceutically acceptable salt thereof. More preferred proteins of the Formula (V) are those wherein: His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser or Pro; Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin or Leu; Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr or Val; Glu at position 105 is replaced with Gin; Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; Gly at position 111 is replaced with Asp; or Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Sex , Thr, Gly, Gin, Val or Leu. Other preferred proteins of the Formula (V) are those wherein: His at position 97 is replaced with Ser or Pro; Trp at position 100 is replaced with Ala, Gly, Gin, Val, lie, or Leu; Ala at position 101 is replaced with Thr; or Trp at position 138 is replaced with Ala, lie, Gly, Gin, Val or Leu. Yet still additional preferred proteins of the Formula (V) are those wherein: His at position 97 is replaced with Ser or Pro; Trp at position 100 is replaced with Ala, Gin or Leu; Ala at position 101 is replaced with Thr; or Trp at position 138 is replaced with Gin. Most preferred species of Formula (V) include species of SEQ ID NO: 16 and 17: (SEQ ID NO:16) 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Ala Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly Cys (SEQ ID NO:17) 5 10 15 Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 100 105 110 His Leu Pro Gin Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly Cys Most significantly, the present invention provides an intermediate, which leads to a very efficient conversion to the biologically active protein conformation of the obesity protein. Therefore, by preparing the intermediate of the present invention, the teriary structure of the biologically active protein is formed first. Thus, allowing almost quantitative conversion of the disulfide bonds. The formation of S-S linked dimer or other multimers is minimized. The protein of the present invention is prepared by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods. Preferably, a protein of Formula (III), or SEQ ID NOs:ll, 12, 13, or 14 is prepared by recombinant synthesis. Recombinant methods are preferred if a high yield is desired. The basic steps in the recombinant production of protein include: a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the protein, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein. a. Gene Construction Synthetic genes, the in vitro or vivo transcription and translation of which will result in the production of the protein may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode the proteins. In the preferred practice of the invention, synthesis is achieved by recombinant DNA technology. Methodology of synthetic gene construction is well known in the art. For example, see Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y., Vol. .68 . , pgs. 109-151. The DNA sequence corresponding to the synthetic protein gene may be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) . It may desirable in some applications to modify the coding sequence of the protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein facilitating the controlled excision of the signal peptide from the fusion protein construct. Techniques for making substitutional mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis. The mutations that might be made in the DNA encoding the present anti-obesity proteins must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See DeBoer et al., EP 75,444A (1983) . The gene encoding the protein may also be created by using polymerase chain reaction (PCR) . The template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue. Such methodologies 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 obesity proteins may be made either by direct expression or as fusion protein comprising the protein followed by enzymatic or chemical cleavage. A variety of peptidases (e.g. trypsin) which cleave a polypeptide at specific sites or digest the peptides from the amino (e.g. diaminopeptidase) or carboxy termini of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi- synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., Carter P., Site Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Soc, Washington, D.C. (1990) . c. Vector Construction Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are digested by restriction enzymes, tailored, and religated to form the plasmids required. To effect the translation of the desired protein, one inserts the engineered synthetic DNA sequence in any of a plethora of appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases. A synthetic coding sequence is designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification plasmids. The isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by techniques well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the protein. In general, plasmid vectors are used which contain promoters and control sequences which are derived from species compatible with the host cell. The vector ordinarily carries a replication site as well as marker sequences which are capable of providing phenotypic selection in transformed cells. For example, E__ coli is typically transformed using pBR322, a plasmid derived from an Ε__ coli species (Bolivar, et al. , Gene 2 : 95 (1977)) . Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA technology. The desired coding sequence is inserted into an expression vector in the proper orientation to be transcribed and translated from a promoter and ribosome binding site, both of which should be functional in the host cell. An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No. 5,304,493, the teachings of which are herein incorporated 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 with restriction enzymes Ndel and BamHI. The genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette. d. Procaryotic expression In general, procaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention. For example, E__ coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include E__ coli B and E__ coli X1776 (ATCC No. 31537) . These examples are illustrative rather than limiting. Also, prokaryotes are used for expression of recombinant proteins. The aforementioned strains, as well as E. coli 3110 (prototrophic, ATCC No. 27325) , bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia arcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the β-lactamase (vector PGX2907 [ATCC 39344] contains the replicon and β-lactamase gene) and lactose promoter systems (Chang et al., Nature, 275:615 (1978); and Goeddel et al. , Nature 281:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC- 37282) . However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein. The following preparations are presented to further illustrate the preparation of the proteins described herein. Preparation 1 A DNA sequence encoding the protein of SEQ ID NO:14 with a Met-Arg N-terminal extension was obtained using standard PCR methodology. A forward primer (5'-GG GG CAT ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC, SEQ ID NO:18) and a reverse primer (5'-GG GG GGATC CTA TTA GCA CCC GG.G AGA CAG GTC CAG CTG CCA CAA CAT, SEQ ID NO:19) was used to amplify sequences from a human fat cell library (commercially available from CLONETECH) . The PCR product is cloned into PCR-Script (available from STRATAGENE) and sequenced. Preparation 2 Vector Construction A plasmid containing the DNA sequence encoding the desired protein is constructed to include Ndel and BamHI restriction sites. The plasmid carrying the cloned PCR product is digested with Ndel and BamHI restriction enzymes. The small ~ 450bp fragment is gel-purified and ligated into the vector pRB182 from which the coding sequence for A-C-B proinsulin is deleted. The ligation products are transformed into E. coli DH10B (commercially available from GIBCO-BRL) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 μg/mL of tetracycline are analyzed. Plasmid DNA is isolated, digested with Ndel and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ~ 450bp Ndel to BamHI fragment are kept. E. coli B BL21 (DE3) (commercially available from NOVOGEN) are transformed with this second plasmid expression suitable for culture for protein production. The techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al. (1988) Molecular Clόninσ: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Current Protocols in Molecular Bioloσy (1989) and supplements. The techniques involved in the transformation of E. coli cells used in the preferred practice of the invention as exemplified herein are well known in the art. The precise conditions under which the transformed !___ coli cells are cultured is dependent on the nature of the !__. coli host cell line and the expression or cloning vectors employed. For example, vectors which incorporate thermoinducible promoter-operator regions, such as the cl857 thermoinducible lambda-phage promoter-operator region, require a temperature shift from about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis. In the preferred embodiment of the invention E. coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, E. coli K12 L201, L687, L693, L507, L640, L641, L695, L814 (E. coli B) . The transformed host cells are then plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for a time and temperature appropriate to the host cell line employed. When expressed in a high-level bacterial expression systems, the protein often aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Kreuger et al., in Protein Foldinσ. Gierasch and King, eds., pgs 136-142 (1990), American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C. The inclusion bodies comprising the obesity protein are solubilized in a denaturant at a concentration sufficient to solubilize the protein, preferably in about 6 to about 8 M urea at a pH from about pH 7 to about pH 12, more preferably 8 to 12. Most preferably, the inclusion bodies are solubilized in about 6 to 7 M urea at a pH of about 8 to 10. The desired protein concentration is about 0.1 mg/mL to the solubility of the protein in the solution; more preferably 0.1 to 50 mg/mL and most preferably 0.5 mg/mL to 5.0 mg/mL. Under these conditions the protein is denatured. Denatured protein molecules regain their native conformation when the renaturation is carried out under carefully controlled conditions. However, renaturation into the biologically active conformation is not a quantitative process; a multitude of non-functional species and conformations of the molecule may be formed. The present invention provides a key intermediate in the renaturation. The claimed intermediate is prepared by adding about 1 to 100 mM, preferably 1 to 20 mM and most preferably 1 to 10 mM, of a thiol reducing reagent containing a free -SH that is operable in a disulfide interchange, preferably, cysteine, cystamine, BME and the like. Preferred thiol reagents are cysteine and cysteamine. The claimed intermediate forms in about 1 minute to 24 hours. The intermediate can be purified by filtration, chromatography or other conventional methodology. Because the intermediate is in the correct tertiary structure (native conformation) , the intermediate can be used as a biologically active therapeutic agent and may offer advantages in efficacy or onset of action. However, the intermediate is preferably converted to the biologically active proteins of Formula III. Most unexpectedly, conversion of the intermediate to the biologically active protein is highly efficient with little or no precipitation of aggregated protein and with minimal formation of covalent dimers or higher order multimers. Protecting the cysteine residues of the protein by the formation of mixed disulfides and thus forming the intermediate, allows for the formation of the tertiary structure largely independent of disulfide bond formation. This condition favors the intramolecular disulfide bond formation in the monomeric biologically active protein. The intermediate is converted to the biologically active protein of the Formula II by reducing the concentration of the thiol reducing reagent and denaturant concentration of the solution to effect disulfide bond formation. The reduction of thiol and denaturant may be carried by dilution, dialysis, diafiltration, or other techniques appreciated in the art. Preferably, after solubilization of the inclusion bodies and formation of the intermediate, the solution is diluted to a protein concentration about 0.05 mg/mL to about 5.0 mg/mL and about 1 to 20 mM thiol and then dialyzed or diafiltered. The buffer used for dialysis or diafiltration is preferably PBS (phosphate buffered saline with about 5 to about 10 mM phosphate and 50 to 500 mM NaCl) at a pH of about 7.0 to 12.0 and more preferably 7.5 to 9.0. Other suitable buffers include, but are not limited to, 4- (2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) , or tris (hydroxymethyl)aminomethane (TRIS) . Preferably, the thiol reducing reagent and denaturant concentration is reduced by diafiltration or dialysis against PBS or about 5 to 10 mM TRIS. The conversion from the intermediate to the biologically active conformation is most remarkable — approaching quantitative conversion. Thus, by passing through the intermediate, the biologically active protein of the Formula II is produced in high yield. The formation of the intermediate also allows the fold to be conducted at higher protein concentrations than a fold carried out directly from the free -SH. The preferred range includes 0.05 to 5 mg/mL, preferably 0.1 to 3 mg/mL, and most preferably 1.0 to 2 mg/mL. A higher concentration during the fold translates into lower volumes (smaller tanks) and less downstream processing. The fold may be carried out in the absence of glycerol or other agent added to prevent protein aggregation. The ability to fold at large scale in the absence of such agents is significant because such agents, particularly glycerol, must be removed in downstream purification. Thus, by processing through the intermediate, the present invention further provides an efficient process of preparing proteins of the Formula II: (a) Solubilizing obesity protein inclusion bodies in a solution comprising: a denaturant at a concentration sufficient to solubilize the protein; and a thiol reducing reagent at a concentration of 1 to 100 mM at a pH from about 7 to about 12; (b) Reducing the thiol and denaturant concentration of the solution to effect disulfide bond formation. Preferably, inclusion bodies are solubilized by the addition of about 6 to 8 M urea and about 3 to 7 mM cysteine in a 8 to 12 mM Tris buffer at about pH 8 to 12 and more preferably at a pH of about 8 to 10. Under these conditions the mixed disulfide intermediate forms and is optionally purified by filtration and/or chromatography. Significantly, the efficiency of the formation of the single intra-chain disulfide from the intermediate is increased by the adding additional thiol reducing reagent prior to dilution, diafiltration or dialysis. Preferably, thiol is added so that it is in molar excess — preferably 1 to 6000 fold, more preferably 3 to 6000 fold, excess. Most preferably, 5 mM thiol, preferably cysteine, is added. Once converted to the biologically active protein, the protein is purified from the reaction mixture by techniques appreciated in the art such as ion exchange chromatography, size exclusion chromatography, reverse phase chromatography, and the like. The intermediate of Formula (I) is stable in the presence or absence of denaturant. The intermediate is soluble in PBS suggesting proper tertiary structure formation. If desired the intermediate may be purified by techniques known in the art and including size exclusion, ion exchange, reversed phase chromatography. The intermediate was characterized on HPLC. A chromatogram for representative intermediates of the claimed invention are presented in Figure 1. Furthermore, the intermediate is identified on SDS-PAGE (sodium dodecyl-sulphate- polyacrylamide gel electrophoresis as a slower migrating species than the biologically active protein. The following examples are presented to further illustrate the invention described herein. The scope of the present invention is not to be construed as merely consisting of the following examples. Comparative Example 1 Protein of SEQ ID NO:14 wherein Xaa at position 28 is Gin and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules were solubilized in 6 M guanidine-HCl, 10 mM sodium acetate (pH 5.0), 1 mM DTT for 1 hour at room temperature. The mixed disulfide of the present invention was not detectable under these conditions. The cysteine of the obesity protein is protonated under these conditions to form (-SH) . Solubilized protein was clarified by centrifugation and renaturation of the clarified protein solution was initiated by dilution (fold dilution = 1:1500) into 20 % glycerol, 5 mM sodium acetate (pH 5.0), 5 mM CaCl 2 to a final protein concentration of 0.025 mg/mL with thorough mixing. The solution became hazy and protein aggregation was noted shortly after dilution. The dilute protein solution was allowed to stand without mixing at room temperature for 8 hours, and the pH of the solution was raised to 8.68 by addition of solid Tris base to 10 mM. The solution was clarified by centrifugation and analyzed by SDS-PAGE under non-reducing conditions, reverse phase HPLC and ESI-mass spectroscopy. Analysis indicated an overall recovery of 68 % of the protein of which 7 % was covalent dimer yielding a 63 % recovery of monomeric protein and a 32 % loss of protein. Comparative Example 2 Protein of SEQ ID NO:14 wherein Xaa at position 28 is Gin and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules were solubilized in 6 M guanidine-HCl, 10 mM sodium acetate (pH 4.5), 1 mM DTT at a protein concentration of 1.3 mg/ L for 1 hour at room temperature. The mixed disulfide of the present invention was not detectable under these conditions. The cysteine is protonated under these conditions to form (-SH) . Solubilized protein was clarified by centrifugation and renaturation of the clarified protein solution was initiated by dilution (fold dilution = 1:55) into 20 % glycerol, 20 mM Tris (pH 8.4), 2.5 mM CaCl 2 to a final protein concentration of 0.025 mg/mL with thorough mixing. The dilute protein solution was allowed to stand without mixing at room temperature for 18 hours. The solution was clarified by centrifugation and analyzed by SDS-PAGE under non-reducing conditions, reverse phase HPLC and ESI-mass spectroscopy. Analysis indicated a recovery of >95 % of the protein of which 16 % was covalent dimer yielding a recovery of monomeric protein of 80 %. Example 1 Protein of SEQ ID NO:14 wherein Xaa at position 28 is Gin and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules were solubilized in 8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine at a protein concentration of 0.1 mg/mL. Renaturation of the protein solution was initiated by dialysis against PBS to remove excess denaturant and cysteine. The solution was clarified by centrifugation and analyzed by SDS-PAGE under non- reducing conditions, reverse phase HPLC and ESI-mass spectroscopy. Analysis indicated a recovery of >95 % of the protein of which < 1 % was covalent dimer yielding an overall recovery of monomeric protein of 94 %. Example 2 Protein of SEQ ID NO:11 wherein Xaa at position 28 is Gin with a Met-Asp N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules constituted the starting material for further purification. The granules were suspended in buffer A (8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine) and found to be soluble in this buffer at high concentrations (up to 40 mg protein/mL) . Solubilized protein was clarified either by centrifugation or by filtration. The protein migrated as a doublet band on nonreducing SDS-PAGE gels and as a single band on reducing SDS-PAGE gels. This is due to the presence of some protein with an internal disulfide bond and some protein with the cysteine residues present as mixed disulfides with the cysteine from the buffer. The protein was initially purified by DEAE anion exchange chromatography in the presence of buffer A. Protein bound to the DEAE resin was eluted with a NaCl gradient to 0.250 mM. Nonreducing SDS-PAGE analysis of fractions indicated that most of the contaminating proteins were present in the leading edge of the main Ob peak. Conservative pooling of the DEAE fractions resulted in relatively pure Ob protein for renaturation. Refolding of the protein was initiated by dilution of the protein to 0.1 mg/mL in PBS. The example was repeated by dilution into buffer A and removing the denaturant and thiol by dialysis into PBS. The protein remained soluble after the dialysis and migrated as a single band on nonreducing SDS-PAGE. Reduction of the protein resulted in a single band with slightly slower mobility on SDS-PAGE indicating that the disulfide bond was completely formed during the renaturation process. Final purification of the protein was achieved by size exclusion chromatography (Superdex 75 column in PBS) purified Ob protein migrated as a single band on SDS-PAGE, yielded a single N-terminal amino acid sequence, which was confirmed by ESI-mass spec. Example 3 A protein of SEQ ID NO:12 was prepared in a manner analogous to Example 1. Example 4 A protein of SEQ ID NO:13 may be prepared in a manner analogous to Example 1. Example 5 Incubation of the partially purified SEQ ID NO:14 with a Met-Asp N-terminal extension in 8 M urea, 10 mM tris, 5 mM cysteine pH 8.0 for 48 hours at 4° C resulted in a mixture of forms of the protein. Nonreducing SDS-PAGE indicated that approximately half of the protein had an internal disulfide bond while the other half did not. Analysis of the protein solution with the sulfhydryl reagent DTNB indicated that no free sulfhydryl was present in solution. This indicates that the cysteine in solution had formed mixed disulfides with the cysteine in the Ob protein. Thus the intermediate in the folding of the biologically active Ob protein had been trapped. An aliquot of this solution was diluted to 0.1 mg/mL in PBS. A second aliquot was diluted to 0.1 mg/mL in PBS with 20 mM DTT. Both diluted samples were then dialyzed against a 10,000 fold excess of PBS for 24 hours. Following dialysis the samples were analyzed by nonreducing SDS-PAGE. The sample diluted into PBS contained both Ob protein with the internal disulfide bond and Ob protein without the internal disulfide (soluble intermediate) . The sample diluted into PBS/DTT contained only Ob protein with the internal disulfide bond. Once formed, the mixed disulfide between Ob and cysteine is stable even after removal of denaturant. The mixed disulfide Ob protein is soluble in PBS suggesting proper secondary structure formation. Addition of excess thiol regent stimulates disulfide exchange and gradual removal of the thiol regent favors formation of the Ob molecule with the internal disulfide bond. Example 6 Protein of Formula (III) (SEQ ID NO:l) wherein Xaa at position 28 is Gin and Trp at position 100 is replaced with Glu and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules constituted the starting material for further purification. The inclusion bodies were solubilized in 8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine; and the mixed disulfide protein was purified by anion exchange chromatography. Purified protein was incubated in Buffer A for 48 hours at 4 C. Confirmation that the cysteine had formed a mixed disulfide (Ri is SCH2C(COOH) (NH2) ) , was carried out using 10 M DTNB in 0.1 M Tris at pH 8 (measuring the absence of free -SH) . Renaturation of the solubilized protein was initiated by dilution of the protein to 0.1 mg/mL with 8 M urea, 10 mM Tris at pH 8.0 and dialysis initiated against PBS. After 24 hours, a 1 mL sample was eluted over a C18 HPLC at 1 mL/minute with a 30-70% CH3CN linear gradient. Two peaks were collected that migrated as two separate bands on SDS PAGE gel in non-reducing buffer. The early eluting peak migrated slower on SDS PAGE, and the mass expected for the claimed mixed disulfide containing intermediates was confirmed by ESI mass spectroscopy. The later eluting peak migrated faster on SDS PAGE. The mass expected for intra- disulfide containing protein of Formula II was confirmed. The analysis confirmed that protein formed the mixed disulfide intermediate of the present invention prior to conversion to the disulfide containing protein. Example 7 Protein of Formula (III) (SEQ ID NO:l) wherein Xaa at position 28 is Gin and Trp at position 100 is replaced with Gin and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules constituted the starting material for further purification. The granules were dissolved in 8 M urea, 10 mM Tris (pH 8.0) , 5 mM cysteine. Renaturation of the protein was initiated by dilution of the protein to 0.1 mg/mL and dialysis against PBS to remove excess denaturant and cysteine. Example 8 Protein of Formula (III) (SEQ ID NO:l) wherein Xaa at position 28 is Gin and Trp at position 100 is replaced with Ala and having a Met-Arg N-terminus extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules constituted the starting material for further purification. The granules were suspended in 8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine. Renaturation of the protein was initiated by dilution of the protein to 0.1 mg/mL and dialysis against PBS to remove excess denaturant and cysteine. N-terminal Met-Arg dipeptide is removed by the using dipeptidylaminopeptidase (dDAP) by techniques appreciated in the art. The protein purified by cation exchange chromatography. Example 9 Protein of Formula (III) (SEQ ID NO:l) wherein Xaa at position 28 is Gin and Trp at position 100 is replaced with Ala and having a Met-Arg N-terminal extension was produced as granules (inclusion bodies) . The granules were isolated by a standard procedure using differential centrifugation. These granules constituted the starting material for further purification. The granules were solubilized by the addition of 7 M urea and 5 mM cysteine in a 10 mM Tris buffer at around pH 8. The solubilized preparation was then clarified by filtration. Under these conditions the cysteine residues of the protein are present as a combination of reduced and cysteinyl mixed disulfides. The urea solubilized mixed disulfide intermediate was purified by anion exchange chromatography on a Big Bead Q- Sepharose column (Pharmacia Fine Chemicals) in 7 M urea, 10 mM Tris, 5 mM cysteine at approximately pH 8. The product is eluted by a linear gradient in NaCl. Additional cysteine is added to the anion exchange purified intermediate, and the pH adjusted to approximately pH 9. The intermediate is then diluted to approximately 2 mg/mL with 7 M urea. Folding and formation of the single intra-chain disulfide bond is accomplished by the removal of urea and cysteine using membrane ultrafiltration/diafiltration against 10 mM Tris at around pH 9. Ultrafiltration membrane with a nominal molecular weight cut-off of 10,000 daltons were used. The N-terminal Met-Arg dipeptide is removed by the using dipeptidylaminopeptidase (dDAP) by techniques appreciated in the art. The protein is administered in a dose between about 1 and 1000 μg/kg. A preferred dose is from about 10 to 100 μg/kg of active compound. A typical daily dose for an adult human is from about 0.5 to 100 mg. In practicing this method, compounds of the Formula (I) can be administered in a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time. The amount per administered dose or the total amount administered will be determined by the physician and depend on such factors as the nature and severity of the disease, the age and general health of the patient and the tolerance of the patient to the compound. The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since they are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention. SEQUENCE LISTING CD GENERAL INFORMATION: (i) APPLICANT: Hale, John E. MacKellar, Warren C. ( ii ) TITLE OF INVENTION: Obesity Protein Intermediates an d Their Preparation and Use (iii) NUMBER OF SEQUENCES: 19 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Eli Lilly and C ompany (B) STREET: Lilly Corporate C en t er (C) CITY: Indianapolis (D) STATE: Indiana (E) COUNTRY: United States (F) ZIP: 46285 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (Vi CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: ( Vϋi ) ATTORNEY /AGENT INFORMATION : (A) NAME: Caltrider , Steven P . (B) REGISTRATION NUMBER: 36 , 467 (C) REFERENCE/DOCKET NUMBER: X-10059 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 317-276-0757 (B) TELEFAX: 317-277-1917 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESΞ: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent " (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr 1 5 10 15 lie Val Thr Arg lie Asn Asp lie Ser His Thr Xaa Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent;- ■ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser 85 90 95 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 49 amino acids (B) TYPE: amino acid (C) ΞTRANDEDNESΞ: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( Xi ) SEQUENCE DESCRIPTION: SEQ ID NO:3: His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 1 5 10 15 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 20 25 30 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu ser Pro 35 40 45 Gly (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gly Ser His Met 1 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Met Gly Ser Ser His His His His His His Ser Ser Gly Lou Val Pro 1 5 10 15 Arg Gly Ser His Met 20 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Leu Glu Lys Arg Glu Ala Glu Ala 1 5 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Glu Ala Glu Ala 1 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Glu Lys Arg 1 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser Pro 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ( i) SEQUENCE DESCRIPTION: SEQ ID NO:10: Gly Ser Pro 1 2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95 His Leu Pro Gin Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Gin Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Val Pro He Trp Arg Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Ser Asp He Ser His Met Gin Ser Val Ser Ser 20 25 30 Lys Gin Arg Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Val 35 40 45 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala He Tyr Gin Gin He 50 55 60 Leu Thr Ser Leu Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys 85 90 95 Pro Leu Pro Gin Ala Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly 100 105 HO Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ala Leu Gin Asp Met Leu Arg Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Val Pro He Cys Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 20 25 30 Lys Gin Arg Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Leu 35 40 45 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala He Tyr Gin Gin He 50 55 60 Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arq Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser Cys 85 90 95 Pro Leu Pro Gin Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val 100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arq 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Arq Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 140 (D) OTHER INFORMATION: /note= "Xaa at position 140 is Gin or absen ." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 20 25 30 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 35 40 45 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 50 55 60 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 65 70 75 80 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 85 90 95 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 100 105 110 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 115 120 125 Ile Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser Ser 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID N0:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95 His Leu Pro Gin Alε Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95 His Leu Pro Gin Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGGGCATATG AGGGTACCTA TCCAGAAAGT CCAGGATGAC A 41 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA ( i) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGGGGGATCC TATTAGCACC CGGGAGACAG GTCCAGCTGC CACAACAT 48

Claims: We claim: 1. A properly folded protein of the Formula ( I ) : 146 ( i : wherein : A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof ; B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein; Rl and R2 are independently H or in conjunction with the sulfur to which it is bound forms a mixed disulfide; provided that both i and R2 are not H . 2 . A protein of Claim 1 , wherein A is a polypeptide of the formula : ( SEQ ID NO : 2 ) 5 10 15 R3 - Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser 35 40 45 Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro 50 55 60 He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 65 70 75 He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp 80 85 90 95 Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser ; wherein: R3 is absent, Met, Met-R4, or a leader sequence; R4 is any amino acid except Pro; Xaa at position 28 is Gin or absent; Gin at position 4 is optionally replaced with Glu; Gin at position 7 is optionally replaced with Glu; Asn at position 22 is optionally replaced with Gin or Asp; Thr at position 27 is optionally replaced with Ala; Xaa at position 28 is optionally replaced with Glu; Gin at position 34 is optionally replaced with Glu; Met at position 54 is optionally replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Gin at position 56 is optionally replaced with Glu; Gin at position 62 is optionally replaced with Glu; Gin at position 63 is optionally replaced with Glu; Met at position 68 is optionally replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Asn at position 72 is optionally replaced with Gin, Glu, or Asp; Gin at position 75 is optionally replaced with Glu; Ser at position 77 is optionally replaced with Ala; Asn at position 78 is optionally replaced with Gin or Asp; or Asn at position 82 is optionally replaced with Gin or Asp. 3. A protein of Claim 2, wherein A is a polypeptide of the formula: (SEQ ID NO:2) 5 10 15 R3 - Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser 35 40 45 Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro 50 55 60 He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 65 70 75 He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp 80 85 90 95 Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser ; wherein: Xaa at position 28 is Gin or absent. 4. A protein of Claim 2 wherein B is a polypeptide of the Formula: (SEQ ID N0:3) 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly ; wherein: His at position 97 is optionally replaced with Gin, Asn, Ala, Gly, Ser, or Pro; Trp at position 100 is optionally replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; Ala at position 101 is optionally replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; Ser at position 102 is optionally replaced with Arg; Gly at position 103 is optionally replaced with Ala; Glu at position 105 is optionally replaced with Gin; Thr at position 106 is optionally replaced with Lys or Ser; Leu at position 107 is optionally replaced with Pro; Asp at position 108 is optionally replaced with Glu; Gly at position 111 is optionally replaced with Asp; Gly at position 118 is optionally replaced with Leu; Gin at position 130 is optionally replaced with Glu; Gin at position 134 is optionally replaced with Glu; Met at position 136 is optionally replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Trp at position 138 is optionally replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or Gin at position 139 is optionally replaced with Glu. 5. A protein of Claim 4 wherein B is a polypeptide of the Formula: (SEQ ID NO:3) 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly . 6. A protein of Claim 4 wherein B is polypeptide of the Formula: (SEQ ID NO:3) 100 105 110 His Leu Pro Ala Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly wherein: Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu. 7. A protein of any one of Claims 2 through 6 wherein R3 is a leader sequence selected from the group consisting of : Gly-Ser-His-Met (SEQ ID NO:4); Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu- Val-Pro-Arg-Gly-Ser-His-Met (SEQ ID NO:5) ; Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO:6) ; Glu-Ala-Glu-Ala (SEQ ID NO:7) ; Leu-Glu-Lys-Arg- (SEQ ID NO:8); Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu- Val-Pro-Arg-Gly-Ser-Pro (SEQ ID NO:9) ; or Gly-Ser-Pro- (SEQ ID NO:10) . 8. A protein of any one of Claims 2 through 6 wherein R3 is a leader sequence selected from the group consisting of: Gly-Ser-His-Met (SEQ ID NO:4); Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu- Val-Pro-Arg-Gly-Ser-His-Met (SEQ ID NO:5); Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO:6); Glu-Ala-Glu-Ala (SEQ ID NO:7) ; Leu-Glu-Lys-Arg- (SEQ ID NO:8) ; Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu- Val-Pro-Arg-Gly-Ser-Pro (SEQ ID NO:9); or Gly-Ser-Pro- (SEQ ID NO:10) . 9. A protein of any one of Claims 2 through 6 wherein R3 is Met Arg. 10. A process of preparing the protein of any one of Claims 1 through 9, which comprises, mixing an obesity protein or analog thereof with a solution comprising a denaturant and a thiol reducing reagent at a concentration of about 1 to 100 mM thiol at a pH from about 7 to about 12. 11. A process of preparing the protein of any one of Claims 1 through 9, which comprises, mixing an obesity protein or analog thereof with a solution comprising a denaturant and a thiol reducing reagent at a concentration of about 1 to 100 iriM thiol at a pH from about 8 to about 12. 12. A process of preparing a biologically active protein of the Formula (II) : 146 (ID wherein: A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof; B is a polypeptide consisting essentially of amino acid residues 96 to 145 of the protein; which comprises: (a) Solubilizing obesity protein inclusion bodies in a solution comprising: a denaturant at a concentration sufficient to solubilize the protein; and a thiol at a concentration of about 1 to 100 mM at a pH from about 7 to about 12; (b) Reducing the thiol and denaturant concentration of the solution to effect disulfide bond formation . 13 . A process of Claim 12 , wherein : A is of the formula : (SEQ ID NO : 2 ) 5 10 15 R 3 - Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys 20 25 30 Thr He Val Thr Arg He Asn Asp He Ser His Thr Xaa Ser Val Ser 35 40 45 Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro 50 55 60 He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 65 70 75 He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp 80 85 90 95 Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser ; wherein: R3 is absent, Met, Met-R4, or a leader sequence; R4 is any amino acid except Pro; and Xaa at position 28 is Gin or absent. 14. A process of Claim 13 wherein B is of the Formula: (SEQ ID NO:3) 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly . 15. A process of Claim 13 wherein B is of the Formula: (SEQ ID NO:3) 100 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 Gly ; Wherein: Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu. 16. A process of any one of Claims 12 through 15, wherein reducing the concentration of thiol and denaturing reagent is carried out by dialysis. 17. A process of any one of Claims 12 through 15, wherein reducing the concentration of thiol and denaturing reagent is carried out by diafiltration. 18. A process of any one of Claims 12 through 15, wherein reducing the concentration of thiol and denaturing reagent is carried out by dilution. 19. A process of preparing a biologically active protein of the Formula (II) : 146 (II) wherein: A is a polypeptide consisting essentially of amino acid residues 1 to 95 of an obesity protein or analog thereof; B is a polypeptide consisting essentially of amino acid residues 97 to 145 of the protein or analog; which comprises: (a) Solubilizing obesity protein inclusion bodies in a solution comprising: a denaturant at a concentration sufficient to solubilize the protein, and a thiol reducing agent at a concentration of 1 to 100 mM, at a pH from about 7 to about 12 ; (b) Adjusting the concentration of the solution td about 0.05 mg/mL to 5.0 mg/mL protein by adding about 1 to 20 mM thiol reducing agent; (c) Reducing the thiol and denaturant concentration of the solution to effect disulfide bond formation. 20. A protein whenever prepared by a process according to any one of claims 10 through 19.

Inventors: HALE JOHN EDWARD, MACKELLAR WARREN CAMERON, HALE, JOHN, EDWARD, MACKELLAR, WARREN, CAMERON
Applicants: HALE JOHN EDWARD, LILLY CO ELI, MACKELLAR WARREN CAMERON, ELI LILLY AND COMPANY, HALE, JOHN, EDWARD, MACKELLAR, WARREN, CAMERON
Application year: 1996
Main classification: C07K1
IPC codes: A23J3, A61K38, C07K1, C07K5, C07K7, C07K14, C12N15, C12P21, C12R1