PROTEIN CRYSTALS, THEIR PREPARATION AND USE

Abstract: The present invention provides crystals of Ob protein, a process for their preparation, and methods for their use. The crystals provide a stable, solid form of the Ob protein that is suitable for X-ray studies, for purification and recovery during manufacture of the Ob protein, and for use as a pharmaceutical for treating obesity and conditions associated with obesity.

Description: PROTEIN CRYSTALS, THEIR PREPARATION AND USE Field of the Invention This invention relates to Ob proteins . More particularly to the preparation of crystals of Ob protein in a form and size suitable for X-ray studies, bulk manufacture, and for pharmaceutical use. Background of the Invention Physiologists have postulated for years that, when a mammal overeats, the resulting excess fat signals to the brain that the body is obese, which, in turn, causes the body to eat less and burn more fuel [Hervey, G. R., Nature 227:629-31 (1969)]. This "feedback" model is supported by parabiotic experiments, which implicate a circulating hormone controlling adiposity. The ob/ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Zhang, Y. and co-workers published the positional cloning of the mouse gene linked with this condition [Zhang, Y., et al . Nature 372:425-432 (1994)]. This report disclosed a gene coding for a 167 amino acid protein with a 21 amino acid signal peptide that is exclusively expressed in adipose tissue. The rat obese gene was subsequently cloned and expressed [Murakami, T., et al., Biochem. Biophys . Res . Com . 209:944- 952 (1995)] . The protein, which is apparently encoded by the o gene, is now speculated to be the adiposity regulating hormone. The porcine and bovine obese gene and protein [Hsiung, H. M., et al . , European Patent Publication ( hereafter, "EP") 0743321 (20 November 1996)] and a primate obese gene and protein [Basinski, M. B., et al, EP 0744408 ( 27 November 1996)] have been previously disclosed. Novel Ob proteins having biological and physical stability properties superior to native obesity proteins are known - 2 - [Basinski, M. B., et al . , WO 96/23515 (8 November 1996); and Basinski, M. B., et al . , WO 96/23517 (8 November 1996)]. S tudies in ob/ob mice have demonstrated that the administration of these proteins causes a reduction in body weight and a correction of the diabetic state. Despite these recent developments relating to the Ob protein, a process for preparing crystals has remained elusive. Until the present invention, the combination of the many parameters that influence nucleation and crystal growth were unknown. The present invention provides crystals of Ob protein, the process for preparation, and use. The crystals provide a stable, solid form of the Ob protein that is suitable for X-ray studies, bulk manufacture, and use as a pharmaceutical. Brief Summary of the Invention The present invention provides crystals of Ob protein, their preparation and use. The invention further provides a process for preparing said crystals, which comprises: mixing a protein solution comprising an Ob protein with a precipitant solution comprising an anion selected from the group consisting of citrate, tartrate, and formate. The invention further provides a process of purifying an Ob protein, which comprises crystallizing said protein to form crystals and isolating said crystals. The invention further provides a pharmaceutical formulation, which comprises crystals of Ob protein, together with one or more pharmaceutically acceptable diluents, carriers, or excipients therefor. The invention further provides a method of treating obesity or those conditions associated with obesity, in a mammal in need thereof, which comprises administering to said mammal an Ob protein crystal. - 3 - Detailed Description of the Invention For purposes of the present invention, as disclosed and claimed herein, the following terms and abbreviations are defined as follows: ACES -- an abbreviation for 2- ( [2-amino-2- oxoethyl] amino) ethanesulfonic acid. ADA -- an abbreviation for N- (carbamoylmethyl) iminodiacetic acid. AMP -- an abbreviation for 2-amino-2-methyl-1- propanol. AMPSO -- an abbreviation for 3- ( [1, l-dimethyl-2- hydroxyethyl] amino) -2-hydroxypropanesulfonic acid. BES -- an abbreviation for N,N-bis(2- hydroxyethyl) --aminoethanesulfonic acid. BICINE -- an abbreviation for N,N-bis(2- hydroxyethyl) glycine . BIS-TRIS -- an abbreviation for 2-bis(2- hydroxyet yl) amino-2- (hydroxymethyl) -1, 3-propanediol . BIS-TRIS-PROPANE -- an abbreviation for 1,3- bis (tris [hydroxymethyl] methylamino) propane . CAPS -- an abbreviation for 3- (eyelohexylamino) -1- propanesulfonic acid. CAPSO -- an abbreviation for 3- (eyelohexylamino) - 2-hydroxy-1-propanesulfonic acid. CHES -- an abbreviation for 2- (N- cyclohexylamino) ethanesulfonic acid. Crystal -- refers to the ordered and three dimensionally periodic internal hexagonal structure. Crystallizing -- refers to the process of preparing crystals. DIPSO -- an abbreviation for 3- (N,N-bis [2- hydroxyethyl] amino) -2-hydroxypropanesulfonic acid. EPPS -- an abbreviation for N-(2- hydroxyethyDpiperazine-N' (3-propanesulfonic acid) . HEPES -- an abbreviation for N-(2- hydroxyethyDpiperazine-N 1 - (2-ethanesulfonic acid) . - A - HEPPSO -- an abbreviation for N- (2- hydroxyethyDpiperazine-N 1 - (2-hydroxypropanesulfonic acid) . MES -- an abbreviation for 2- (N- morpholino) ethanesulfonic acid. MOPSO -- an abbreviation for 3- (N-morpholino) -2- hydroxypropanesulfonic acid. MOPS -- an abbreviation for 3- (N- morpholino)propanesulfonic acid. Obesity protein or Ob 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 sequences of several obesity proteins have been published, including, the mouse obesity protein and human obesity protein [Zhang, Y., et al . Nature 372:425-432 (1994)], the rat obesity protein [Murakami, T. et al., Biochem . Biophys . Res . Comm . 209:944-952 (1995)], the porcine and bovine obese gene and protein [Hsiung, H. M., et al . , EP 0743321 (20 November 1996)] and a primate obese gene and protein [Basinski, M. B., et al , EP 0744408 (27 November 1996)] . These disclosures are incorporated herein by reference . The numbering of amino acids in the present specification is consecutively from the amino terminus of the full length, mature 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 Val at position 1 (such as a methionine) , that do not affect the novel and basic characteristics of the invention are included in the definition of Ob protein. The term Ob protein also includes various obesity protein analogs, some of which have enhanced biological activity or stability. An analog of Ob protein is a native Ob protein having one or more amino acid substitutions, preferably less than five, and most preferably less than three substitutions. Ob protein, therefore, includes obesity protein analogs of Formula (I) that have biological and physical stability properties superior to native obesity proteins [Basinski, M. B., et al . , WO 96/23515 (8 November 1996); and Basinski, M. B., et al . , WO 96/23517 (8 November 1996)] : 5 10 15 Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr 20 25 30 lie 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 lie Pro Gly Leu His Pro lie 50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin lie 65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val lie 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 (SEQ ID NO:l) (I) 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; - 6 - 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 . The terms obesity protein and Ob protein, as used herein, also encompass the obesity protein analogs of the Formula (II) : 5 10 15 Xaa Xaa He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Xaa 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 Xaa Xaa 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 Xaa 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 Xaa Gin Leu Asp Leu Ser Pro 145 Gly Cys (SEQ ID NO: 2) (II) wherein: Xaa at position 1 is Val or absent; Xaa at position 2 is Pro or absent; Xaa at position 22 is Asn or Ser; Xaa at position 28 is Gin or absent; Xaa at position 72 is Asn, Gin, Glu or Asp; Xaa at position 73 is Val or Met; Xaa at position 100 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; Xaa at position 138 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions : Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg; Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg; lie at position 3 is replaced with Glu, Asp, Arg, Lys, or His; - 8 - Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His; Val at position 36 is replaced with Glu, Asp, Arg, Lys, or His; Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 42 is replaced with Glu, Asp, Arg, Lys, or His; Pro at position 43 is replaced with Glu, Asp, Arg, Lys, or His; Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His; His at position 46 is replaced with Glu, Asp, Arg, or Lys; Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 48 is replaced with Glu, Asp, Arg, Lys, or His; Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His; Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 74 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Phe at position 92 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof. Finally, the terms obesity protein and Ob protein, as used herein, also include the obesity protein analogs of Formula (III) : - 9 - 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 (SEQ ID NO:3) (III) wherein: Xaa at position 22 is Asn or Ser; Xaa at position 28 is Gin or absent; Xaa at position 72 in Asn, Gin, Glu, or Asp; Xaa at position 73 is Val or Met; said protein having at least one of the following substitutions : Trp at position 100 is replaced with Glu, Asp, His, Lys, or Arg; Trp at position 138 is replaced with Glu, Asp, His, Lys, or Arg; or a pharmaceutically acceptable salt thereof . Ob gene -- refers to any nucleic acid sequence that hybridizes with, and is at least 50% homologous to, preferably 70% homologous to, and most preferably 80% homologous to one of the native Ob gene sequences disclosed by Zhang, Y., et al . [Nature 372:425-432 (1994)] or Murakami, T., et al . , [Biochem . Biophys . Res . Co . 209:944- - 10 - 95 2 ( 1 995)] . The Ob gene product is expressed specifically in adipose tissue and regulates energy balance. PIPES -- an abbreviation for 1,4- piperazinediethanesulfonic acid. POPSO -- an abbreviation for piperazine-N,N'bis (2- hydroxypropanesulfonic acid) . TAPS -- an abbreviation for ( [2-hydroxy-1, 1- bis (hydroxymethyl) ethyl] amino) -1-propanesulfonic acid. TAPSO -- an abbreviation for 3-(N- tris [hydroxymethyl] methylamino) -2-hydroxypropanesulfonic acid. TEA -- an abbreviation for triethanolamine . TES -- an abbreviation for N- tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid. 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. TRICINE -- an abbreviation for N- tris (hydroxymethyl) methylglycine . TRIS or Tris -- an abbreviation for tris (hydroxymethyl) -aminomethane. TRIZMA -- an abbreviation for tris (hydroxymethyl) aminomethane (TRIS) . The abbreviations used for amino acids herein are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. § 1.822 (b) (2) (1993) . Unless otherwise indicated the amino acids are in the L configuration. As noted, the present invention provides crystals of an Ob protein, their preparation and use. The conditions described herein to crystallize the Ob protein are sensitive - 11 - to the protein and precipitant anion selected. Preferred Ob proteins are those modified to reduce physical aggregation such as those of Formula (I) , (II) , or (III) . Ob proteins having a higher propensity to aggregate, such as the human Ob protein, crystallize much more slowly and under a narrower range of conditions described herein. Optimizing such conditions for the desired Ob protein and anion selected is well within the skill of the art. The invention provides a process for preparing Ob protein crystals, which comprises mixing a protein solution comprising an Ob protein with a precipitant solution comprising an anion selected from the group consisting of citrate, tartrate, and formate. The concentration of protein that can be used to crystallize is limited by the solubility of the protein. Preferably, the protein concentration is about 1 mg/mL to about 50 mg/mL; more preferably about 2 mg/mL to 20 mg/mL; and most preferably about 2 mg/mL to 10 mg/mL. For obesity protein analogs having increased physical stability in solution, such as those of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO: 4, and especially for the obesity protein analogs of SEQ ID NO: 7-11, for example, further preferred ranges of protein concentration for forming the crystals of the present invention are from about 10 mg/mL to about 100 mg/mL; more preferably from about 15 mg/mL to about 45 mg/mL, and most preferably from about 20 mg/mL to about 40 mg/mL. A precipitant solution comprises an anion selected from the group consisting of citrate, tartrate, and formate. Preferred precipitant anions are tartrate and formate. Citrate is a preferred precipitant anion also, for, among other reasons, its suitability for large-scale manufacturing processes . The concentration of precipitant anion to induce the crystallization is not critical. However, the selection and concentration of anion affects the quality and size of crystal produced. The preferred concentration of precipitant anion is about 1 mM to about 6 M. More preferably, it is 100 mM to 2.5 M. Most preferably, the - 12 - concentration of anion is 0.5 M to 1.5 M, which yields crystals measuring 250 to 500 microns. Other preferred anion concentrations are dependent on the anion and protein selected. Generally, formate is preferably at a concentration of about 1.5 M to 2.5 M and most preferably about 2 M. Tartrate is preferably about 0.3 M to about 1.2 M and most preferably about 0.8 M. Optimizing the anion concentration for particular species of Ob protein and for the anion selected is carried out by techniques appreciated in the art. The cation in the precipitant solution is not critical. Acceptable cations include sodium, potassium, cesium, lithium, and any mixture of the aforementioned cations. Ammonium cations are less preferred and may result in inoperable embodiments depending on the concentration, and protein selected. The process may optionally include additional excipients to facilitate or optimize the crystallization. Such excipients may be added to either the precipitant solution, the protein solution, or both. Such excipients include buffer, salts, surfactants, or organic solvents recognized as being acceptable to the crystallization of proteins . Suitable buffers include sodium acetate, sodium citrate, sodium cacodylate, imidazole, sodium HEPES, TRIS HC1, BIS-TRIS, ADA, ACES, MES, PIPES, MOPSO, BIS-TRIS- PROPANE, BES, MOPS, TES, HEPES, DIPSO, TAPSO, TRIZMA, HEPPSO, POPSO, EPPS, TEA, TRICINE, BICINE, TAPS, AMPSO, CHES, CAPSO, AMP, and CAPS. Salts, such as sodium chloride, in addition to the anion required for crystallization can be present without affecting the crystallization. Surfactants may be optionally added to the crystallization to prevent the protein from aggregating and precipitating prior to nucleation and crystal growth. These additives are particularly important in those Ob proteins having an increased tendency to aggregate such as the human O b protein. Surfactants suitable to facilitate crystallization include Tween 20 (polyoxyethylene (20) sorbitan monolaurate) , Tween 40 (polyoxyethylene (20) sorbitan monopalmitate) , Tween 80 (polyoxyethylene (20) sorbitan monooleate) , and Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers) . An organic solvent may also be optionally added to the crystallization. The organic solvent competes with the protein for water and reduces the dielectric constant of the protein solution enhancing the intermolecular electrostatic interactions of the protein molecules and thereby promotes protein crystallization. Suitable organic solvents include BRIJ 35 (polyoxyethylene (23) lauryl ether) polyethlyene glycol (PEG 400, 1500, 4000, and 8000) , isopropanol, ethanol, t-butanol, and 2-methyl-2, 4-pentanediol . The crystallization is carried out at about pH 6 to about pH 13. More preferably, the crystallization is carried out from about pH 7 to about pH 10, and most preferably from about pH 7.5 to about pH 8.5. The temperature of the crystallization is not critical and can range from about 0°C to about 40°C. Preferably, the crystallization is carried out from about 4°C to about 20°C, and most preferably at about 20°C for certain purposes. A temperature of about 4°C may be preferable for forming larger crystals. Mixing the precipitant solution and the protein solution can be carried out by any of the plethora of recognized techniques including dialysis, vapor diffusion, batch, or interface diffusion. These techniques are described in detail in CRYSTALLIZATION OF NUCLEIC ACIDS AND PROTEINS, Ducruix, A. and Giege, R., Chapter 4, Oxford University Press, New York, 1992, herein incorporated by reference. Preferably, the crystallization is carried out in a batch operation. That is, the protein solution and precipitant solution are mixed. Crystals generally appear within about 10 to 24 hours. - 14 - The crystallization process, particularly, when using the batch method, is useful in purifying Ob protein from a process stream in the manufacture of the protein. Protein crystallization is generally possible only after a substantial degree of purity has been reached. However, surprisingly, the present crystallization may be carried out from very crude mixtures, such as those found in the manufacture of the protein from a recombinant fermentation starting material . The present invention therefore serves as a powerful technique for the isolation of pure obesity proteins in bulk. Thus, the invention provides a process of purifying an Ob protein, which comprises crystallizing said protein to form crystals and isolating said crystals. In addition, the crystals provide a stable, solid state suitable for bulk storage. Ob protein crystals of the present invention are hexagonal in shape, having space group P6 3 , space group number 173 , and the following unit cell dimensions to within ± 2 A: A=B= 87 A; C= 48 A; α =β= 90°; γ = 120°. Preferably, the unit cell dimension are within ± 1 A of: A=B=87.65 A; C=47.64 A; α =β= 90°; γ = 120°; identified in accordance with International TABLES FOR X-RAY CRYSTALLOGRAPHY, VOL. l: SYMMETRY GROUPS, The Kynoch Press, Birmingham, England (1952) . The resolution is better than 2.3 A and solvent content about 65%. From the X-ray crystal structure, it is concluded that the structure of the obesity protein is similar to other proteins having the so-called "long-chain cytokine structure" [Sprang, S. R., et al . . Current Opinion in Structure Biology, 3:815-827 (1993)]. Crystals described herein were prepared using tartrate and formate anions. However, crystals having the above space group and unit cell dimensions may also be prepared with other anions including those selected from the group consisting of citrate, sulfate, phosphate, and chloride. Such crystals are included within the scope of the present invention. The proteins of the present invention are 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, an Ob protein of the present invention is properly folded and is prepared by techniques known in the art and taught, for example, in Hsiung, H. M., et al . , EP 0743321 (20 November 1996); Hale, J. E., et al . , WO 97/00866 (9 January 1997); Basinski, M. B., et al . , WO 96/23515 (8 November 1996); and Basinski, M. B., et al . , WO 96/23517 (8 November 1996) . Each of the aforementioned publications is expressly incorporated herein by reference for enabling the skilled reader to prepare obesity proteins. Preferred embodiments of the present invention are formed of native obesity protein and obesity protein analogs described by Formulas (I) , (II) , or (III) . Particularly preferred for the practice of the present invention are proteins described by Formula (I) . Within this preferred group of proteins are further preferred groups, such as, proteins of Formula (I) , wherein: Asn at position 72 is replaced with Gin, Glu, or Asp; the amino acid at position 100 is Trp or is replaced with Glu or Asp; and the amino acid at position 138 is Trp or is replaced with Glu or Asp; or a pharmaceutically acceptable salt thereof . Another preferred group of proteins for the present crystals are those of Formula (I) , wherein Asn at position 72 is replaced with Asp. Further preferred embodiments are crystals comprised of proteins Formula (II) or (III) . Another group of preferred embodiments is composed of crystals of the native Ob proteins, including murine, porcine, bovine, and human. - 16 - Additional preferred embodiments are crystals of the obesity protein analogs of the Formula (IV) : 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 (SEQ ID NO : 4 ) ( IV) wherein : Xaa at position 28 is Gin or absent ; Xaa at position 72 is Asn or Arg; said protein having at least one substitution selected from the group consisting of : Trp at position 100 is replaced with Glu, Asp , His , Lys , or Arg ; or Trp at position 138 is replaced with Glu, Asp, His, Lys, or Arg; or a pharmaceutically acceptable salt thereof . The most preferred obesity protein analog species for use in the present invention include those obesity protein analogs having amino acid sequences represented by SEQ ID NO: 5 through SEQ ID NO: 11, as given below: - 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 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: 5) 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 (SEQ ID NO: 6) - 18 - 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 Asp 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: 7) 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 Glu 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: 8) - 19 - 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 Asp 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 Asp 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: 9) 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 Asp 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 Asp 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: 10) 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 Asp 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 Asp 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: 11) 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. Example 1 Crystals of the Protein of SEQ ID NO: 7 by Vapor Diffusion A protein solution comprising a protein of SEQ ID NO: 7, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with a precipitant solution comprising 0.8 M sodium potassium tartrate, 0.1 M TAPS-Na, pH 8.5 ("tartrate/TAPS solution"), at 20°C. The tartrate/TAPS solution (2 μL) was mixed with 2 μL of the protein solution on silicon-coated glass cover slips. These cover slips were inverted and sealed over wells containing 1 mL of the tartrate/TAPS solution. Crystals appeared within 12-24 hours. These crystals were in the form of hexagonal rods. - 21 - Example 2 Crystals of the Protein of SEQ ID NO: 5 by Vapor Diffusion A protein of SEQ ID NO: 5 was crystallized using the vapor diffusion technique in a manner substantially analogous to Example 1 to give crystals in 3-5 days. These crystals were in the form of hexagonal rods. Example 3 Crystals of the Protein of SEQ ID NO: 8 by Vapor Diffusion A protein of SEQ ID NO: 8 was crystallized in a manner substantially analogous to Example 1 to give crystals within 12-24 hours. These crystals were in the form of hexagonal rods . Example 4 Crystals of the Protein of SEQ ID NO: 7 by a Batch Method A protein of SEQ ID NO: 7, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with 0.8 M sodium potassium tartrate, 0.1 M TAPS-Na, pH 8.5 at 20°C. The tartrate/TAPS solution (5 μL) was mixed with 5 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted and sealed over an otherwise empty crystallization chamber. Crystals appeared within 12-24 hours. These crystals were in the form of hexagonal rods. Example 5 Crystals of the Protein of SEQ ID NO: 8 by a Batch Method A protein of SEQ ID NO: 8 was crystallized in a manner substantially analogous to Example 4 to give crystals within 12-24 hours. The crystals were in the form of hexagonal rods . Example 6 Crystals of the Protein of SEQ ID NO: 7 by Vapor Diffusion A protein solution comprising a protein of SEQ ID NO: 7, previously concentrated to 7 mg/mL as determined by - 22 - optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with a precipitant solution comprising 1.8 M sodium formate, 0.1 M TRIS, pH 9.0 ("1.8 M formate/TRIS solution") at 21°C. The 1.8 M formate/TRIS solution (2 μL) was mixed with 2 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted and sealed over wells containing 1 mL of the formate/TRIS solution. Crystals appeared within 12-24 hours. These crystals were in the form of hexagonal rods. Example 7 Crystals of the Protein of SEQ ID NO: 5 by Vapor Diffusion A protein solution comprising a protein of SEQ ID NO: 5, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with a precipitant solution comprising 2.0 M sodium formate, 0.1 M TRIS, pH 9.0 ("2.0 M formate/TRIS solution") at 4°C. The 2.0 M formate/TRIS solution (2 μL) was mixed with 2 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted and sealed over wells containing 1 mL of the 2.0 M formate/TRIS solution. Crystals appeared within 12-24 hours. These crystals were in the form of hexagonal rods. Example 8 Crystals of the Protein of SEQ ID NO: 8 by Vapor Diffusion A protein solution comprising a protein of SEQ ID NO: 8, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with a precipitant solution comprising 1.8 M sodium formate, 0.1 M AMPSO, pH 8.5 ("1.8 M formate/AMPSO solution") at 21°C. The 1.8 M formate/AMPSO solution (2 μL) was mixed with 2 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted and sealed over wells containing 1 L of the 1.8 M formate/AMPSO solution. Crystals appeared within 12-24 hours. These crystals were in the form of hexagonal rods. - 23 - Example 9 Crystals of the Protein of SEQ ID NO: 7 by a Batch Method A protein of SEQ ID NO: 7, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with 1.0 M sodium formate, 0.1 M TRIS, pH 9.0 ("1.0 M formate/TRIS solution") at 21°C. The 1.0 M formate/TRIS solution (5 μL) was mixed with 5 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted and sealed over an otherwise empty crystallization chamber. Crystals in the form of hexagonal rods appeared within 12-24 hours. Example 10 Crystals of the Protein of SEQ ID NO: 5 by a Batch Method A protein solution comprising a protein of SEQ ID NO: 5, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with a precipitant solution comprising 2.0 M sodium formate, 0.1 M TRIS, pH 9.0 ("2.0 M formate/TRIS solution) at 4°C. The 2.0 M formate/TRIS solution (5 μL) was mixed with 5 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted and sealed over an otherwise empty crystallization chamber. Crystals in the form of hexagonal rods appeared within 12-24 hours . Example 11 Crystals of the Protein of SEQ ID NO: 8 by a Batch Method A protein solution comprising a protein of SEQ ID NO: 8, previously concentrated to 7 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with a precipitant solution comprising 1.8 M sodium formate, 0.1 M AMPSO, pH 8.5 ("1.8 M formate/AMPSO solution) at 21°C. The 1.8 M formate/AMPSO solution (5 μL) was mixed with 5 μL of the protein solution on silicon coated glass cover slips. These cover slips were inverted - 24 - and sealed over an otherwise empty crystallization chamber. Crystals in the form of hexagonal rods appeared within 12-24 hours . To further exemplify the full range of conditions described herein Examples 12 through 154 are presented in Table 1. The protein concentration of the protein solution was 6.5-7.0 mg/mL, the protein solution contained 20 mM TRIS buffer, pH 7.5, and the crystallization method was vapor diffusion. In the table are described the buffer species in the precipitant solution (all were at 0.1 M) , the pH of the precipitant solution, the precipitant salt species in the precipitant solution, the concentration of the precipitant salt in the precipitant solution, any additives used and their concentrations, and the temperature at which the crystallizations were carried out. The crystallizations were done in using 24-well plates. Equal volumes of the protein solution and the precipitant solution were mixed and applied to cover slips, as described in the examples above. In all other ways, the vapor diffusion experiments were carried out as described above. The designation of a concentration range (e.g. Example 29 was carried out at 0.6 to 1.0 M) indicates the concentration was varied by 0.1 M from 0.6 to 1.0 M and found to be operable at all concentrations. Examples 12 through 27 were carried out with the protein of SEQ ID NO: 8. Examples 28 through 70 were carried out with a protein of SEQ ID NO: 7. Examples 71 through 112 were carried out with the protein of SEQ ID NO: 8. Examples 113 through 154 were carried out with a protein of SEQ ID NO: 10, using a protein solution at 7 mg/mL. Under each of the conditions described, crystals in the form of hexagonal rods were observed by the unaided eye. 25 - Table 1 26 - 27 - 28 - Example 155 Crystals of the Protein of SEQ ID NO: 9 A protein solution comprising a protein of SEQ ID NO: 9, previously concentrated to 20 mg/mL as determined by optical density, in 20 mM TRIS buffer and 200 mM NaCl, pH 7.5, was crystallized with various precipitant solutions comprising 2.0 M to 3.6 M sodium formate, 0.1 M TRIS, pH 8.5 ("formate/TRIS solution"), at 4°C to 20°C. The formate/TRIS solution (2.5 μL) was mixed with 2.5 μL of the protein solution on silicon-coated glass cover slips. These cover slips were inverted and sealed over wells containing 1 mL of the formate/TRIS solution. Crystals appeared under all precipitant concentrations and temperatures within 24 hours and continued to grow for several days. Size of the crystals range from about 50 μm X 50 μm X 300 μm to very large crystals having dimensions of 150 μm X 150 μm X 150 μm. A temperature of about 4°C may be preferable for forming larger crystals. Example 156 Crystals of the Protein of SEQ ID NO: 9 A protein solution comprising a protein of SEQ ID NO: 9, previously concentrated to 40 mg/mL as determined by optical density, in 20 mM TRIS buffer, pH 7.5, was crystallized with various precipitant solutions comprising 2.0 M to 3.6 M sodium formate, 0.1 M TRIS, pH 8.5 ( "formate/TRIS solution"), at 4°C to 20°C. The formate/TRIS solution (2.5 μL) was mixed with 2.5 μL of the protein - 29 - solution on silicon-coated glass cover slips. These cover slips were inverted and sealed over wells containing 1 mL of the formate/TRIS solution. Crystals appeared under all precipitant concentrations and temperatures within 24 hours and continued to grow for several days. Size of the crystals range from about 50 μm X 50 μm X 300 μm to very large crystals having dimensions of 150 μm X 150 μm X 150 μm. A temperature of about 4°C may be preferable for forming larger crystals. The Ob protein crystal is also a useful drug delivery form. The crystals may be administered by a variety of means including orally, pulmonary, or by injection. The preferred dosage administered is a dose between about 1 and 1000 μg/kg. A more 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 the method of the present invention, crystals 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 condition, the age and general health of the patient and the tolerance of the patient to the Ob protein crystal. Significant advantages of administering the protein as a crystal include fewer side effects, improved efficacy, and longer duration of action. The instant invention further provides pharmaceutical formulations comprising crystals of the present invention. The crystals can be formulated for parenteral administration for the therapeutic or prophylactic treatment of obesity. For example, the crystals of the present invention can be admixed with conventional pharmaceutical carriers and excipients. The compositions comprising the claimed crystals contain from about 0.1 to 95% by weight of the active protein, and more generally from about 10 to 30%. Furthermore, the crystals - 30 - of the present invention may be administered alone or in combination with other anti-obesity agents or agents useful in treating diabetes. For intravenous (i.v.) use, the formulation is administered in commonly-used intravenous fluids, for example, by infusion. Such fluids, for example, physiological saline, Ringer's solution, or 5% dextrose solution can be used. For intramuscular preparations, a sterile formulation of the crystals of the present invention can be administered as a suspension in a pharmaceutical diluent such as pyrogen-free water (distilled) , physiological saline, or 5% glucose solution. A suitable formulation of the crystals of the present invention may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e . g. , an ester of a long-chain fatty acid such as ethyl oleate. Pharmaceutically acceptable preservatives such as an alkylparaben, particularly methylparaben, ethylparaben, propylparaben, or butylparaben or chlorobutanol are preferably added to the formulation to allow multi-dose use. The formulation may also contain an isotonicity agent, which is an agent that is tolerated physiologically and imparts a suitable tonicity to the formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other possible isotonicity agents include salts, e.g., NaCl, dextrose, mannitol, and lactose. Glycerin is the preferred isotonicity agent. The concentration of the isotonicity agent is in the range known in the art for parenteral formulations, and for glycerin, is preferably about 16 mg/mL to about 25 mg/mL. The formulation may also contain a pharmaceutically acceptable buffering compound to control the pH at a desired level. The pH is ideally such as to be acceptable to the patient upon administration, yet one at which the formulation is sufficiently stable, both physically and chemically. Preferably, the pH is controlled from a mildly acidic pH to a mildly basic pH, such as, between about pH 5 and pH 9. More preferably, the pH is between about pH 6 and pH 8. Buffering compounds, such as, without limitation, citrate, acetate, phosphate, TRIS, or a basic amino acid, such as, lysine or arginine, are known to be pharmaceutically acceptable in these pH ranges. Other pharmacologically acceptable buffers for buffering at pH in these ranges are known in the art. The selection and concentration of buffer is known in the art. The ability of the present Ob protein crystals to treat obesity is demonstrated in vivo as follows: Biological Testing Parabiotic experiments suggest that a protein is released by peripheral adipose tissue and that the protein is able to control body weight gain in normal, as well as obese mice [Coleman, D. L., Diabetologia 14:141-148 (1978)]. Therefore, the most closely related biological test is to inject the test article by any of several routes of administration, e . g. , intravenous (i.v.), subcutaneous (s.c), intraperitoneal (i.p.), or by minipump or cannula, and then to monitor food and water consumption, body weight gain, plasma chemistry or hormones (glucose, insulin, ACTH, corticosterone, GH, T4) over various time periods. Suitable test animals include normal mice (ICR, etc.) and obese mice ( ob/ob, Avy/a, KK-Ay, tubby, fat). The ob/ob mouse model of obesity and diabetes is generally accepted in the art as being indicative of the obesity condition. Controls for non-specific effects for these injections are done using vehicle with or without the active agent of similar composition in the same animal monitoring the same parameters or the active agent itself in animals that are thought to lack the receptor (db/db mice, fa/fa or cp/cp rats) . Proteins demonstrating activity in these models will demonstrate similar activity in other mammals, particularly humans. 32 Since the target tissue is expected to be the hypothalamus where food intake and lipogenic state are regulated, a similar model is to inject the test article directly into the brain, for example, by injection via lateral or third ventricles (i.e.v.) or directly into specific hypothalamic nuclei, such as, the arcuate, paraventricular, or perifornical nuclei. The same parameters as above could be measured, or the release of neurotransmitters that are known to regulate feeding or metabolism could be monitored (e.g. NPY, galanin, norepinephrine, dopamine, β-endorphin release) . Similar studies are accomplished in vitro using isolated hypothalamic tissue in a perifusion or tissue bath system. In this situation, the release of neurotransmitters or electrophysiological changes is monitored. Example 157 Testing of a Crystal Suspension of the Protein of SEQ ID NO: 8 in ob/ob Mice Ob/ob mice (50-60 grams, from Jackson Labs) were maintained on Purina 5008 chow and regular lighting (lights on from 4:30 AM until 4:30 PM) . There were 5 mice in each treatment group. They were injected subcutaneously (SC) once daily for four days with the formulations shown below. The mice were weighed daily at 3:30 P.M. Their food and water consumption were determined at the same time. Treatment, as indicated below, was made following weighing, just prior to lights out. The mice were treated once daily for 4 days . - 33 The effects of these treatments on food consumption and cumulative body weight change are illustrated for representative proteins of the present invention in Table 2. Table 2. Effect of SEQ ID NO: 8 on food consumption and cumulative body weight change in ob/ob mice The Ob protein crystals are active in at least one of the above biological tests and are anti-obesity agents. As such, they are useful in treating obesity and those disorders implicated by obesity such as diabetes, cardiovascular disease, and cancer. However, the crystals of obesity proteins are not only useful as pharmaceutical agents, but also are useful for controlling weight for cosmetic purposes in mammals. A cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance. The mammal is not necessarily obese. Such cosmetic use forms part of the present invention. 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. - 34 - SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Briggs, Stephen L Clawson, David K Schevitz, Richard W Wery, Jean-Pierre Zhang, Faming (ii) TITLE OF INVENTION: Protein Crystals, Their Preparation and Use (iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Eli Lilly and Company (B) STREET: Lilly Corporate Center (C) CITY: Indianapolis (D) STATE: Indiana (E) COUNTRY: U.S.A. (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: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: WO N/A (W/ Filing) (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Caltrider, Steven P. (B) REGISTRATION NUMBER: 36,467 (C) REFERENCE/DOCKET NUMBER: X-11066 (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) 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; said protein having at least one of the following substitutions : " (ix) FEATURE: (A) NAME/KEY: Modified-site - 35 - (B) LOCATION: 4 (D) OTHER INFORMATION: /note= "Gin at position 4 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 7 (D) OTHER INFORMATION: /note= "Gin at position 7 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 22 (D) OTHER INFORMATION: /note= "Asn at position 22 is replaced with Gin or Asp;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 27 (D) OTHER INFORMATION: /note= "Thr at position 27 is replaced with Ala;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is replaced with Glu; " ( ix) FEATURE : (A) NAME/KEY: Modified-site (B) LOCATION: 34 (D) OTHER INFORMATION: /note= "Gin at position 34 is replaced with Glu; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 54 (D) OTHER INFORMATION: /note= "Met at position 54 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;" ( ix) FEATURE : (A) NAME/KEY: Modified-site (B) LOCATION: 56 (D) OTHER INFORMATION: /note= "Gin at position 56 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 62 (D) OTHER INFORMATION: /note= "Gin at position 62 is replaced with Glu; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 63 (D) OTHER INFORMATION: /note= "Gin at position 63 is replaced with Glu;" ( ix) FEATURE : (A) NAME/KEY: Modified-site (B) LOCATION: 68 (D) OTHER INFORMATION: /note= "Met at position 68 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;" ( ix) FEATURE : (A) NAME/KEY : Modif ied-site - 36 - (B) LOCATION : 72 (D) OTHER INFORMATION : /note= "Asn at position 72 is replaced with Gin, Glu, or Asp ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 75 (D) OTHER INFORMATION: /note= "Gin at position 75 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 77 (D) OTHER INFORMATION: /note= "Ser at position 77 is replaced with Ala;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 78 (D) OTHER INFORMATION: /note= "Asn at position 78 is replaced with Gin or Asp;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 82 (D) OTHER INFORMATION: /note= "Asn at position 82 is replaced with Gin or Asp;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 97 (D) OTHER INFORMATION: /note= "His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 100 (D) OTHER INFORMATION: /note= "Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu;" ( ix) FEATURE : (A) NAME/KEY: Modified-site (B) LOCATION: 101 (D) OTHER INFORMATION: /note= "Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 102 (D) OTHER INFORMATION: /note= "Ser at position 102 is replaced with Arg;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 103 (D) OTHER INFORMATION: /note= "Gly at position 103 is replaced with Ala;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 105 (D) OTHER INFORMATION: /note= "Glu at position 105 is replaced with Gin;" (ix) FEATURE: - 37 - (A) NAME/KEY: Modified-site (B) LOCATION: 106 (D) OTHER INFORMATION: /note= "Thr at position 106 is replaced with Lys or Ser ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 107 (D) OTHER INFORMATION: /note= "Leu at position 107 is replaced with Pro;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 108 (D) OTHER INFORMATION: /note= "Asp at position 108 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 111 (D) OTHER INFORMATION: /note= "Gly at position 111 is replaced with Asp ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 118 (D) OTHER INFORMATION: /note= "Gly at position 118 is replaced with Leu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 130 (D) OTHER INFORMATION: /note= "Gin at position 130 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 134 (D) OTHER INFORMATION: /note= "Gin at position 134 is replaced with Glu;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 136 (D) OTHER INFORMATION: /note= "Met at position 136 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 138 (D) OTHER INFORMATION: /note= "Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 139 (D) OTHER INFORMATION : /note= "Gin at position 139 is replaced with Glu; or a pharmaceutically acceptable salt thereof . " (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 - 38 - 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 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: 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: 1 (D) OTHER INFORMATION: /note= "Xaa at position 1 is Val or absent ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= "Xaa at position 2 is Pro or absent;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 22 (D) OTHER INFORMATION: /note= "Xaa at position 22 is Asn or Ser; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 72 (D) OTHER INFORMATION: /note= "Xaa at position 72 is Asn, Gin, Glu or absent;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 73 (D) OTHER INFORMATION: /note= "Xaa at position 73 is Val or Met ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 100 (D) OTHER INFORMATION: /note= "Xaa at position 100 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or ..." (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 138 (D) OTHER INFORMATION: /note= "Xaa at position 138 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions : " ( ix) FEATURE : (A) NAME/KEY: Modified- site (B) LOCATION: 1 (D) OTHER INFORMATION: /note= "Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 2 (D) OTHER INFORMATION: /note= "Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /note= "He at position 3 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 30 (D) OTHER INFORMATION: /note= "Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 36 (D) OTHER INFORMATION: /note= "Val at position 36 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 41 (D) OTHER INFORMATION: /note= "Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 42 (D) OTHER INFORMATION: /note= "He at position 42 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site - 40 - (B) LOCATION : 43 (D) OTHER INFORMATION : /note= " Pro at position 43 is replaced with Glu, Asp, Arg , Lys , or His ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 45 (D) OTHER INFORMATION: /note= "Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 46 (D) OTHER INFORMATION: /note= "His at position 46 is replaced with Gly, Asp, Arg, or Lys;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 47 (D) OTHER INFORMATION: /note= "Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 48 (D) OTHER INFORMATION: /note= "He at position 48 is replaced with Gly, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 49 (D) OTHER INFORMATION: /note= "Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 50 (D) OTHER INFORMATION: /note= "Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 74 (D) OTHER INFORMATION: /note= "He at position 74 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 89 (D) OTHER INFORMATION: /note= "Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 92 (D) OTHER INFORMATION: /note= "Phe at position 92 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;" ( ix) FEATURE : (A) NAME/KEY: Modified-site (B) LOCATION: 99 (D) OTHER INFORMATION: /note= "Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or" ( ix) FEATURE : (A) NAME/KEY : Modif ied-site - 41 - (B) LOCATION: 142 (D) OTHER INFORMATION: /note= "Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof . " (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Xaa Xaa He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 . 10 15 He Val Thr Arg He Xaa 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 Xaa Xaa 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 Xaa 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 Xaa Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO: 3: (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: 22 (D) OTHER INFORMATION: /note= "Xaa at position 22 is Asn or Ser;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 72 (D) OTHER INFORMATION: /note= "Xaa at position 72 is Asn, - 42 - Gin, Glu, or Asp; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 73 (D) OTHER INFORMATION: /note= "Xaa at position 73 is Val or Met; said protein having at least one of the following substitutions : " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 100 (D) OTHER INFORMATION: /note= "Trp at position 100 is replaced with Glu, Asp, His, Lys, or Arg;" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 138 (D) OTHER INFORMATION: /note= "Trp at position 138 is replaced with Glu, Asp, His, Lys, or Arg; or a pharmaceutically acceptable salt thereof . " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 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 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: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein - 43 - ( ix) FEATURE : (A) NAME/KEY: Modified-site (B) LOCATION: 28 (D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent ; " (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 72 (D) OTHER INFORMATION: /note= "Xaa at position 72 is Asn or Arg; said protein having at least one substitution selected from the group consisting of:" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 100 (D) OTHER INFORMATION: /note= "Trp at position 100 is replaced with Glu, Asp, His, Lys, or Arg; or" (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 138 (D) OTHER INFORMATION: /note= "Trp at position 138 is replaced with Glu, Asp, His, Lys, or Arg; or a pharmaceutically acceptable salt thereof." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 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 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: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear - 44 - (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 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 Ala 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: 6: (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: 6: 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 - 45 - 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: 7: (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:7: 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 Asp 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: 8: - 46 (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 : 8 : 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 Glu 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: 9: (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: 9: 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 - 47 - 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 Asp 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 Asp 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: 10: (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: 10: 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 Asp 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 Asp 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 - 48 - (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 (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 Gin Ser Val Ser Ser 20 25 30 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp 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 Asp 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 X-11066

Claims: - 49 - We Claim: 1. Crystals of Ob protein. 2. Crystals of Claim 1, wherein the crystals are hexagonal rods having space group P6 3 and space group number 173; having the following unit cell dimensions to within ± 2 A: A=87 A, B=87 A, and C=48 A; and having angles of α=90°, β=90°, and γ=120°. 3. Crystals of Claim 2, wherein the protein is selected from the group consisting of human Ob protein, mouse Ob protein, porcine Ob protein, and bovine Ob protein. 4. Crystals of Claim 2, wherein the protein is of the Formula (I) : 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 (SEQ ID NO : l) ( I ) wherein : Xaa at position 28 is Gin or absent ; - 50 - 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; - 51 - Gin at position 134 is replaced with Glu; Met at position 136 is replaced with methionine sulfoxide, Leu, He, Val, Ala, or Gly; Trp at position 138 is replaced with Ala, Glu, A sp, Asn, Met, He, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or Gin at position 139 is replaced with Glu; or a pharmaceutically acceptable salt thereof. 5. Crystals of Claim 4, wherein Asn at position 72 is replaced with Gin, Glu, or Asp; the amino acid at position 100 is Trp or is replaced with Glu or Asp; and the amino acid at position 138 is Trp or is replaced with Glu or Asp; or a pharmaceutically acceptable salt thereof . 6. Crystals of Claim 2, wherein the protein is of the Formula (II) : 5 10 15 Xaa Xaa He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30 He Val Thr Arg He Xaa 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 Xaa Xaa 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 Xaa 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 Xaa Gin Leu Asp Leu Ser Pro 145 - 52 - Gly Cys (SEQ ID NO: 2) (II) wherein: Xaa at position 1 is Val or absent; Xaa at position 2 is Pro or absent; Xaa at position 22 is Asn or Ser; Xaa at position 28 is Gin or absent; Xaa at position 72 is Asn, Gin, Glu or Asp; Xaa at position 73 is Val or Met; Xaa at position 100 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; Xaa at position 138 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions : Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg; Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg; He at position 3 is replaced with Glu, Asp, Arg, Lys, or His; Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His; Val at position 36 is replaced with Glu, Asp, Arg, Lys, or His; Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His; He at position 42 is replaced with Glu, Asp, Arg, Lys, or His; Pro at position 43 is replaced with Glu, Asp, Arg, Lys, or His; Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His; His at position 46 is replaced with Glu, Asp, Arg, or Lys; Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His; - 53 - He at position 48 is replaced with Glu, Asp, Arg, Lys, or His; Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His; Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His; He at position 74 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Phe at position 92 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof . 7. Crystals of Claim 2, wherein the protein is of Formula (III) : 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 - 54 - Gly Cys (SEQ ID NO: 3) ( III ) wherein: Xaa at position 22 is Asn or Ser; Xaa at position 28 is Gin or absent; Xaa at position 72 in Asn, Gin, Glu, or Asp; Xaa at position 73 is Val or Met; said protein having at least one of the following substitutions : Trp at position 100 is replaced with Glu, Asp, His, Lys, or Arg; Trp at position 138 is replaced with Glu, Asp, His, Lys, or Arg; or a pharmaceutically acceptable salt thereof . 8. A crystal of an obesity protein selected from the group consisting of obesity protein analogs having amino acid sequences represented by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:ll. 9. A process for preparing a crystal of any one of Claims 1 through 8, which comprises mixing a protein solution comprising an Ob protein with a precipitant solution comprising an anion selected from the group consisting of citrate, tartrate, and formate. 10. A process for preparing a crystal of any one of Claims 1 through 8, which comprises mixing a protein solution comprising an Ob protein with a precipitant solution comprising an anion selected from the group consisting of tartrate and formate. 11. A process of purifying an Ob protein, which comprises crystallizing said protein to form crystals, and isolating said crystals . - 55 - 12. A method of treating obesity, or conditions associated with obesity, in a mammal in need thereof, which comprises administering to said mammal a crystal of any one of Claims 1 through 8. 13. A pharmaceutical formulation, comprising, a crystal as claimed in any one of Claims 1 to 8, together with one or more pharmaceutically acceptable diluents, carriers or excipients therefor.

Inventors: BRIGGS STEPHEN L, CLAWSON DAVID K, SCHEVITZ RICHARD W, WERY JEAN-PIERRE, ZHANG FAMING, BRIGGS, STEPHEN, L., CLAWSON, DAVID, K., SCHEVITZ, RICHARD, W., WERY, JEAN-PIERRE, ZHANG, FAMING
Applicants: BRIGGS STEPHEN L, CLAWSON DAVID K, LILLY CO ELI, SCHEVITZ RICHARD W, WERY JEAN PIERRE, ZHANG FAMING, BRIGGS, STEPHEN, L., CLAWSON, DAVID, K., ELI LILLY AND COMPANY, SCHEVITZ, RICHARD, W., WERY, JEAN-PIERRE, ZHANG, FAMING
Application year: 1997
Main classification: A61K38
IPC codes: A61K38, C07K14