Title of Invention	PROCESS TO PRODUCE INSULINOTROPIC PEPTIDES
Abstract	The insulinotropic peptide, exendin-4, is overexpressed in Pichia pastoris using a codon optimized encoding sequence with a optional signal peptide and/or propeptide. The expression of the exogenous gene is driven by the methanol inducible promoter AOX. The purified peptide from the recombinant organism is alpha amidated at the penultimate serine. The production of exendin-4 may also be carried out in bacteria such as E, coli or in yeast such as Saccharomyces cerevisiae. The purified recombinant peptide is useful in formulating pharmaceutical compositions for the treatment of diseases such as diabetes and obesity.

Title of Invention

PROCESS TO PRODUCE INSULINOTROPIC PEPTIDES

Abstract

The insulinotropic peptide, exendin-4, is overexpressed in Pichia pastoris using a codon optimized encoding sequence with a optional signal peptide and/or propeptide. The expression of the exogenous gene is driven by the methanol inducible promoter AOX. The purified peptide from the recombinant organism is alpha amidated at the penultimate serine. The production of exendin-4 may also be carried out in bacteria such as E, coli or in yeast such as Saccharomyces cerevisiae. The purified recombinant peptide is useful in formulating pharmaceutical compositions for the treatment of diseases such as diabetes and obesity.

Full Text	Field of the Invention The present invention provides a method of producing an insulinotropic peptide (e.g., exendin-4) by cloning and expressing the native peptide or an engineered form of the peptide in microorganisms such as yeast. Background of the Invention A number of hormones that lower blood glucose levels are released from the gastrointestinal mucosa in response to the presence and/or absorption of nutrients in the gut. These hormones include gastrin, secretin, glucose-dependent insulinotropic polypeptide (GIF), and glucagon-like peptide-1 (GLP-1). GLP-1 is a proteolytic product of proglucagon, a 180 amino acid peptide which also gives rise to glucagon peptide (Orskov 1992 Diabetologia 35; 701-711; Drucker 1998, Diabetes Al\ 159-169; each of which is incorporated herein by reference). GLP-1 has a number of functions: (i) it is a physiological hormone that enhances the effect on insulin secretion in normal humans and is therefore an incretin hormone; (ii) GLP-1 also lowers glucagon concentrations, slows gastric emptying, stimulates (pro) insulin biosynthesis and enhances insulin sensitivity (Nauck 1997, Horm. Metab. Res, 47; 1253-1258; incorporated herein by reference); (iii) the peptide also enhances the ability of the beta-cells to sense and respond to glucose in subjects with impaired glucose tolerance (Byrne 1998, Eur, J. Clin. Invest. 28; 72-78; incorporated herein by reference); and (iv) the insulinotropic effect of GLP-1 in humans increases the rate of glucose disappearance partly because of increased insulin levels and partly because of enhanced insulin sensitivity (D'Alessio 1994, J. Clin. Invest, 93; 2263-2266; incorporated herein by reference). However, a major pharmacological drawback to native GLP-1 is its short half-life. In humans, GLP-1 is rapidly degraded by dipeptidyl peptidase (DPP-IV) into GLP-1 (9-36) amide, which acts as an endogenous GLP-1 receptor antagonist (Deacon, 1998 Diabetologia 41; 271-278; incorporated herein by reference). Several strategies have been proposed to limit this problem, for example, using inhibitors of DPP-IV, or using DPP-IV-resistant analogues of GLP-1 (Deacon et al, 1998, Diabetes 47; 764-769; Ritzel, 1998 J. Endocrinol. 159; 93-102; US Patent 5545618, Pederson, 1998 Diabetes 47; 1253-1258; each of which is incorporated herein by reference). Exendins, another family of peptides that lower blood glucose levels have some sequence similarity (53%) to GLP-1 (Goke et al, 1993 J. Biol Chem. 268; 19650-19655; incorporated herein by reference). The exendins are found in the venom of the Gila Monster (Raufman 1996 Reg, Peptides, 61; 1-18; incorporated herein by reference) and the Mexican beaded lizard. Exendin-4 found in the venom of the Gila Monster {Heloderma suspectum) is of particular interest. Published PCT international patent application, WO 98/35033, which is incorporated herein by reference, discloses the cDNA encoding proexendin peptide, including exendin and other novel peptides, its isolation, and antibodies which specifically recognize such peptides (Pohl et al 1998, J. Biol Chem, liy, 9778-9784; incorporated herein by reference). Exendin-4 has been shown to be a strong GLP-1 agonist in isolated rat insulinoma cells. This is expected since the His-Ala domain of GLP-1 recognized by DPP-IV is not present in exendin-4, which has the sequence His-Gly instead (Goke et. al, 1993, J. Biol Chem. 268; 19650-19655; incorporated herein by reference). Sequences of GLP-1 and Exendin-4 GLP-l (7-37) HAEGTFTSDVSSYLEGQAAKEFLWVLVKGRG (SEQ ID NO: 1) EXENDIN-4 (1-40) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2) Exendin-4 given systematically lowers blood glucose levels by 40% in diabetic db/db mice (WO 99/07404; incorporated herein by reference). Recently, Grieg et al (Diabetologia 1999 42; 45-50; incorporated herein by reference) has shown a long lasting blood glucose lowering effect of once daily intraperitoneal injections of exendin-4 in diabetic ob/ob mice. U.S. Patent 5,424,286 by Eng, which is incorporated herein by reference, discloses that a considerable portion of the N-terminal sequence is essential in order to preserve insulinotropic activity whereas an N-terminally truncated exendin (exendin-4 (9-39)) has inhibitory properties. The use of exendin-4 and exendin agonists has been proposed for the treatment of diabetes mellitus, reducing gastric motility and delaying gastric emptying, and preventing hyperglycemia (U.S. Patent 5,424,286; WO 98/05351; each of which is incorporated herein by reference) as well as for reducing food intake (WO 98/30231; incorporated herein by reference). However, the number of amino acid residues in a peptide sequence has a dramatic influence on the production costs of peptides made by solid phase synthetic chemistry. The cost of manufacturing a 50-mer peptide is at least 5 times greater than the cost of a 10-mer peptide. Solid phase peptide synthesis also requires the use of corrosive solvents such as TFS and hydrofluoric acids, a number of synthetic steps required to produce the peptide, and subsequent purification of the peptide. Therefore, there is a need for efficiently and cost effectively producing large quantities of biologically active exendin-4 and other insulinotropic peptides for the treatment of these conditions. Summary of the Invention The present invention provides a system for preparing insulinotropic peptides by heterologous gene expression in microorganisms. This system provides for large-scale recombinant production of peptides such as exendin-4. The system provides a way of producing large quantities of biologically active peptides without resorting to solid phase peptide synthesis. The peptides produced using the inventive system may be used in formulating pharmaceutical compositions. The peptides and compositions thereof may be used in treating diabetes mellitus, reducing gastric motility, delaying gastric emptying, preventing hyperglycemia, and reducing appetite and food intake. The present invention provides a method of producing insulinotropic peptide having biological activity, the method comprising steps of: - providing a host cell transformed with a polynucleotide vector encoding the polypeptide with insulinotropic activity; and - growing the host cell under suitable conditions to produce the polypeptide. In producing an insulinotropic peptide by heterologous gene expression in a microorganism, the encoding polynucleotide sequence for the peptide, preferably as part of a vector, is transformed into a host cell of the microorganism. The expression of the gene encoding the insulinotropic peptide is controlled by a promoter (e.g., an inducible promoter or constitutive promoter). Preferably, the promoter is a strong promoter, more preferably a strong inducible promoter, which allows for the production of large quantities of the insulinotropic peptide. The cells transformed with the gene may be fungal (e.^., yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris)). For expression in P, pastoris, the promoter used to drive the expression of the gene is preferably the AUGl promoter, the GAP promoter (a strong constitutive promoter), or the AOXl or A0X2 promoter (a strong inducible promoter); and the vector may be derived from a known or commercially available vector such as pPIC, pPIC3K, pPIC9K, or pHIL-D. For expression in S. cerevisiae, the promoter used may be the CUPI promoter, ADH promoter, the TEFl promoter, or the GAL promoter. The polynucleotide sequence encoding the insulinotropic peptide being expressed may be optimized for expression in the particular organism {e.g., codon usage may be optimized for use in the host microorganism). The polynucleotide sequence may optionally include leader sequences, signal sequences, pre-/pro-peptide sequences, tags for processing, tags for detection or purification, fusion peptides, etc. In certain embodiments, the gene encodes a signal peptide. The signal peptide may direct the processing or secretion of the peptide. In certain embodiments, the signal peptide is the signal peptide of exendin-4 or the signal peptide of mat alpha factor. In certain embodiments, the gene encodes a propeptide, and the propeptide includes the native exendin-4 propeptide or the propeptide of mat alpha factor (see WO 98/35033; Pohl et al,J. Biol Chem. 273:9778-9784, 1998; each of which is incorporated herein by reference). The transformed host cells are selected for the presence of the vector (e.g., antibiotic resistance, ability to grown on media which does not contain a particular nutrient) and/or expression of the exogenous gene encoding the insulinotropic peptide. The selected cells are then grown under conditions suitable for expression of the gene encoding the peptide. In certain embodiments, the medium in which the cells are grown include an agent, such as methanol, which induces the expression of the gene encoding the peptide. In other embodiments, when a constitutive promoter is used, an agent for inducing the expression for the exogenous gene is not necessary. The transformed host cell produces the peptide. Preferably, peptide is properly processed by the host cell. That is, the peptide is properly folded and post-translationally modified. In certain embodiments, the peptide is secreted it into the growth medium. The recombinantly produced peptide is then isolated and purified from the host cells or from the medium, if the peptide has been secreted. The purified peptide may be further modified chemically or enzymatically (e.g-., alpha amidation, cleavage). The isolated, modified, and/or purified peptide may be used in formulating pharmaceutical compositions and treating patients with a particular disease or condition involving glucose metabolism/uptake. The compositions may include insulinotropic peptides of a specific activity greater than the activities seen in peptides produced by other means such as solid phase peptide synthesis. These compositions may be used to treat such endocrine diseases as diabetes mellitus. These compositions may also be used to treat conditions such as reduced gastric motility, delayed gastric emptying, hyperglycemia, and obesity. In one aspect, the insulinotropic peptide, exendin-4, is produced. The peptide may be produced in any microorganism, preferably Pichia pastoris. The production of recombinant exendin-4 in Pichia pastoris involves preparing the expression construct comprising a signal sequence, a pro-peptide, a pre-peptide, and/or a tag, which is followed by the 40 amino acid exendin-4 sequence in tandem. In certain embodiments, the signal sequence comprises the native exendin-4 signal sequence of 23 amino acid or a 19 amino acid alpha factor signal sequence. The propeptide comprises the native exendin-4 propeptide of 24 amino acids or a 66 amino acid propeptide of alpha factor. The tag may aid in the processing of the peptide (Kjeldsen et ah Biotechnol Appl Biochem. 29:79-86, 1999; incorporated herein by reference). In certain embodiments, the tag is a charged ammo acid sequence such as the EAEA spacer described in Example 6. A Pichia codon optimized nucleotide sequence coding for exendin-4 optionally with a signal peptide or propeptide is prepared. The synthetic nucleotide sequence is then cloned into an appropriate vector carrying a selectable marker gene, such as pPIC, pPIC3K, pPIC9K, or pHIL-D. The vector is transformed into Pichia pastoris cells, and transformed cefls carrying the vector are selected. The selected clone is fermented on a large enough scale to produce the desired quantity of exendin-4 peptide. The exendin-4 peptide so produced into the broth need no further processing steps to make it a fully functional peptide unlike that produced in bacterial system (EP 978565 Ohsuye et al Suntory limited; incorporated herein by reference). The peptide is purified from the medium using protein purification techniques known in the art. The exendin-4 peptide is preferably not glycosylated by the Pichia host cells. Lack of glycosylation is also an advantage because the Exendin-4 is fully functional and it leads to a simpler and easier purification process and results in improved yields of the peptide. The purified exendin-4 peptide may be further chemically or enzymatically modified (e,g,, C-terminal alpha amidation). The exendin-4 peptide may be used in pharmaceutical compositions for administration to a subject (e.g., humans). The invention also provides kits and reagents useful in recombinantly producing insulinotropic peptides in microorganisms. These kits may include polynucleotides, nucleotides, vectors, enzymes, cells, buffers, media, vials, plates, Eppendorf tubes, chromatographic materials, instructions, etc. Preferably, the kit is conveniently packaged for use in a laboratory setting. In certain embodiments, Pichia pastoris cells transformed with a vector encoding exendin-4 are included in the kit. In certain embodiments, the kit may also include the materials necessary for purifying the overexpressed peptide. Definitions "Animal": The term animal, as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). An animal may be a transgenic animal. "Effective amount": In general, the "effective amount" of an active agent or the microparticles refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a peptide or pharmaceutical composition may vary depending on such factors as the desired biological endpoint, the peptide to be delivered, the disease or condition being treated, etc, "Peptide" or "protein": According to the present invention, a "peptide" or "protein" comprises a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids {i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification {e.g., alpha amindation), etc. In a preferred embodiment, the modifications of the peptide lead to a more stable peptide {e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide. In certain embodiments, the modifications of the peptide lead to a more biologically active peptide. "Transformation": The term "transformation" as used herein refers to introducing a vector comprising a nucleic acid sequence into a host cell. The vector may mtegrate itselt or a portion or itseii mro me cnromosome oi me ceiis, or me vector may exist as a self-replicating extrachromosomal vector. Integration is considered in some embodiments to be advantageous since the nucleic acid is more likely to be stably maintained in the host cell. In other embodiments, an extrachromosomal vector is desired because each host cell can contain multiple copies of the vector. Brief Description of the Drawing Figure 7 is a schematic showing modified versions of the exendin-4 gene including signal peptide and propeptide sequences. These modified versions of exendin-4 may be used in the Pichia pastoris expression system. Detailed Description of Certain Preferred Embodiments of ttie Invention The inventive system for recombinantly producing insulinotropic peptides is particularly usefiil in preparing large quantities of biologically active peptide for use in pharmaceutical compositions or for research purposes. The invention provides methods of preparing the polynucleotide and recombinant cells for producing the peptide as well as methods of recombinantly producing the peptide itself The invention also includes polynucleotide sequences, vectors, cells, and kits usefiil in practicing the inventive methods, and pharmaceutical compositions and methods of using the recombinantly produced peptides. As described above, recombinantly expressing an insulinotropic peptide in a host microorganism involves preparing a polynucleotide with the peptide-encoding sequence, introducing this sequence into a vector, transforming cells of the microorganism with the vector, and selecting for cells with the vector. These selected cells are then grown under suitable conditions to produce the insulinotropic peptide, which is purified from the cells or from the medium in which the cells were growing. The purified peptide is then used in pharmaceutical compositions to treat such diseases as diabetes or obesity. Polynucleotide The production of a recombinant protein/peptide begins with the preparation of a polynucleotide sequence that encodes the insulinotropic peptide, or a modified version of the peptide, to be produced. Preferably, the peptide has been characterized sufficiently that the primary sequence of the peptide is known. The sequence may include sections of the peptide that are cleaved from the final product such as signal sequences, tags, fusion peptides or proteins, and/or pre-/pro-sequences. In certain instances, the gene or cDNA encoding the peptide is known in the art or has been cloned so that the primary sequence of the peptide has been deduced from the nucleotide sequence. In other embodiments, the primary sequence of the peptide has been determined {e.g., by Edman degradation), but the gene or cDNA encoding the peptide has not been cloned. The sequence of the gene, cDNA, and/or peptide may be available in the scientific literature or in a genetic database such as GenBank. The present invention focuses on the production of insulinotropic peptides such as glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1 (GLP-1), and exendin family members (e.g., exendin-4); therefore, the sequences of these peptides, as well as variants, homologs, mutants, allelic variants, isotypes, polymorphisms, etc., thereof, are particularly useful. In certain embodiments, exendin-4 is expressed. Exendin-4 has the primary amino acid sequence (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2)). The amidated form of exendin-4 with the C-terminal glycine residue removed may also be produced and has the sequence ((HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 3)). In certain embodiments, the exendin-4 peptide or its amidated form is not glycosylated. The pro-exendin gene product has the primary amino acid sequence (MKIILWLCVFGLFLATLFPISWOMPVESGLSSEDSASSESFASKIKRHGEGTFT SDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 4)). The underlined residues indicate the pro-sequence. In designing the peptide sequence for expression in a recombinant microorganism, the peptide sequence may be altered from the wild type sequence. The alteration may be a mutation of one or more of the amino acids found in the peptide. The alteration may be a deletion or addition of an amino acid or plurality of amino acids. In certain embodiments, a sequence of amino acids is added to the peptide for recombinant production. Such a sequence can be added at the ends or in the middle of the peptide; however, preferably, the sequence is added at the C-terminus or N-terminus of the peptide. For example, a signal sequence may be added, preferably at the N-terminus of the peptide. A propeptide sequence may be added. A prepeptide sequence may be added. The peptide may be fused with another protein or peptide. A cleavage site may also be added. In certain embodiments, the resulting peptide sequence contains at least a contiguous 10,15, 20, 25, 30, or 35 amino acid sequence at least 75%, 80%, 90%, 95%, 98%, or 100% homologous to the wild type sequence. In certain embodiments, there is at least a 20 amino acid sequence that is at least 95% homologous to the wild type sequence. Once the peptide sequence has been designed, the corresponding polynucleotide which encodes the engineered peptide sequence can be prepared. As will be appreciated by one of skill in this art, due to the redundancy in the genetic code, more than one polynucleotide sequence can encode a peptide. Any polynucleotide sequence that encodes the engineered peptide can be used in the recombinant production of the peptide. However, in certain embodiments, the polynucleotide sequence is optimized for expression in a particular organism. That is, the codon(s) commonly used by the organism to encode a particular amino acid are used in generating the encoding polynucleotide sequence. For example, if one were producing a peptide in Pichiapastoris, it would be preferable to use the codons in the genetic code more commonly used by this organism in expressing its own proteins (see Table 1 below, and U.S. Patent 5,827,684, incorporated herein by reference). For expression in S. cerevisiae, the codon usage table for S. cerevisiae may be used (see ftp://genome-ftp.stanford.edu/pub/codon/ysc.gene.cod; incorporated herein by reference). In certain embodiments, the most often used codon for an amino acid is not used but instead the second or third most frequently used codon is used in constructing the polynucleotide sequence. As would be appreciated by one of skill in this art, codon usage depends upon the organism being used to express the peptide. Therefore, a polynucleotide sequence used to express a protein in one organism may not be optimal for expression in a different organism. In a particularly preferred embodiment, the invention provides a polynucleotide sequence encoding exendin-4, which has been optimized for expression in Pichia pastoris. The sequence is as follows: CTC GAG AAA AGA CAT GGA GAA GGA ACA TTT ACA TCT GAT TTG TCT AAA CAA ATG GAA GAA GAA GCT GTT AGA TTG TTT ATT GAA TGG TTG AAA AAC GGA GGA CCA TCT TCT GGA GCT CCA CCA CCA TCT GGA TAA GAA TTC (SEQ ID NO: 5). In certain embodiments, the polynucleotide sequence is at least 90%, 95%, 97%, 99%, or 100% homologous to the optimized sequence over the entire sequence. In other embodiments, at least a 25, 50, 75, or 100 nucleotide sequence is at least 90%, 95%, 97%, 99%, or 100% homologous to the analogous section of the optimized sequence. As would be appreciated by one of skill in this art a polynucleotide encoding a signal sequence, propeptide sequence, prepeptide sequence, or tag may be added to the sequence. In certain embodiments, the polynucleotide encoding the signal sequence, propeptide sequence, prepeptide sequence, or tag is provided by the vector the sequence is subcloned into. In such a case, the peptide encoding DNA sequence is inserted in frame into the open reading frame of the vector. In other embodiments, the exendin-4 encoding polynucleotide is optimized for expression in S. cerevisiae. These sequence may also include signal sequences or prepeptide sequences. Vector In order to express a polynucleotide in a cell, the polynucleotide is typically incorporated into a vector. The vector may be prepared using any techniques known in the art (please see, Molecular Cloning: A Laboratory Manual^ 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); the treatise, Methods in Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Ausubel et al Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); each of which is incorporated herein by reference). The vector may be a plasmid, cosmid, chromosome, artificial chromosome, virus, phase, naked piece of DNA, etc. In certain embodiments, the vector is a plasmid. Preferably, the plasmid can be amplified in E. coti. The vector may be DNA or RNA based. The vector may be double-stranded DNA or RNA, or single-stranded DNA or RNA. The vector may include other sequences, for example, selection markers (e.g., antibiotic resistance genes, biosynthetic genes (e.g., URA3, LEU2, TRPl, HIS3)), origins of replication (e.g., 2\|im, ARS (autonomously replicating sequence), ori), housekeeping genes, restriction sites, etc. In certain embodiments, the vector includes a cloning site (i.e., polylinker) for inserting the gene to be expressed. The cloning site may be flanked by a promoter, enhancer sequences, ribosomal binding site, transcription start site, TATA box, termination site, etc. In certain embodiments, a constitutive promoter is used to express the peptide-encoding gene. In other embodiments, an inducible promoter is used to express the peptide-encoding gene. Typically the promoter is species specific. Examples of promoters for use in P. pastoris include the AOXl, A0X2, AUGl, and GAP promoters. In certain embodiments, the AOXl promoter is used for expression in P. pastoris. Example of promoters useful in S. cerevisiae include the CUPl, ADH, TEFl, and GAL promoters. The vector is typically designed for use in a particular organism. In other embodiments, the vector is designed for use in Saccharomyces cerevisiae. In yet other embodiments, the vector is designed for use in Pichia pastoris. Examples of plasmid vectors for use in Pichia pastoris include pPIC, pPIC3K, pPIC9K, and pHIL- D. In certain embodiments, the vector is commercially available. In certain embodiments, the plasmid vector include a polynucleotide encoding exendin-4 for expression in P. pastoris. Preferably, the vector is a plasmid derived from pPIC, pPIC3K, pPIC9K, and pHIL-D. Cells The vector encoding the peptide to be expressed is used to transform a cell. The cell may be a fungal cell, in particular a yeast cell. In certain embodiments, the cell is a S. cerevisiae cell. In certain other embodiments, the cell is a Pichia pastoris cell. The cells may be obtained commercially or may be obtained from depositories such as the ATCC. In certain embodiments, the cells are wild type cells. In other embodiments, the cells have been engineered. In other embodiments, the cells have been altered by the hand of man. The vector is used to transform the cell by any means known in the art (please see. Molecular Cloning: A Laboratory Manual^ 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); the treatise. Methods in Enzymology (Academic Press, Inc., N.Y.); Ausubel et al. Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); each of which is incorporated herein by reference). Techniques for getting the vector into the cell include electroporation, calcium phosphate precipitation, transformation of competent cells, viral infection, phage infection, etc. In certain embodiments, electroporation is used to get the vector into the cell. After the gene encoding the peptide to be expressed is transformed into the cells, the cells with the gene may be selected. This selection may be done by growing the cells in a particular medium which requires the expression of a gene on the vector. For example, the vector may encode an antibiotic resistance gene and the medium may contain the antibiotic. Cells which not have the vector will be killed by the antibiotic whereas cells with the antibiotic resistance gene will be able to grow. In certain embodiments, the cells are grown in media lacking an essential nutrient. The vector confers the ability to grow on such media; therefore, cells with the vector grow while cells without the vector cannot divide. In certain embodiments, expression of the peptide may be determined directly by immunoassay or enzymatic assay. After cells containing the vector with the gene to be expressed are selected, they may be grown in medium under suitable conditions to produce the desired peptide. The medium used to grow the cells, aeration, temperature, and the time course for growing the cells and inducing the cells to produce the peptide is determined by one of skill in this art or can be determined empirically. In growing recombinant ftingi (e.g., P. pastoris), the cells are grown between 22°C-32'C, preferably between 26°C-30'C. The aeration of the fermentation culture maintains dissolved oxygen above 20%, 25%, or 30%. Preferably, dissolved oxygen is maintained above 30%. In certain embodiments, an agent may be added to the growth media to induce the expression of the gene. In other embodiments, the gene is expressed constitutively without the need for such an inducing agent. The peptide may be harvested from the culture after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10 days, or 14 days. In certain embodiments, the expression level of the insulinotropic peptide exceeds 100 mg/L. In other embodiments, the expression level exceeds 200 mg/L. In yet other embodiments, the expression level exceeds 300 mg/L. The organism chosen for expression of the peptide should correctly fold and modify the peptide so that the peptide has the same or greater biological activity than the peptide derived from its natural source. The peptide may be phosphorylated, glycosylated, cleaved, etc. by the machinery of the host cell. In certain embodiments, the sites for glycosylation on the peptide are not glycosylated by the host cells. Preferably, the peptide is not glycosylated at any sites. The lack of glycosylation may lead to easier purification and/or improved yields of the peptide. In certain embodiments, signal peptides, pro-peptide sequences, and pre-peptide sequences are cleaved from the peptide. In other embodiments, the peptide is processed in the cell and then secreted into the media the cells are growing in. After the peptide has been harvested from the fermentation, the peptide is purified. The peptide may be purified from the cells, or if the peptide is secreted, the peptide may be purified from the media. If the peptide is purified from cells, the cells are lysed, and the peptide is purified from the cell lysate. The peptide may be purified using any methods known in the art including ammonium sulfate precipitation, column chromatography (e.g. ion exchange chromatography, size exclusion chromatography, affinity chromatography), FPLC, HPLC {e,g., reverse phase, normal phase), e^c. The peptide is purified to the desired level of purity. In certain embodiments, the final peptide is greater than 90% pure, 95% pure, 98% pure, or 99% pure, preferably greater than 98% pure. The purified peptide may be used as is, or the peptide may be further modified. In certain embodiments, the peptide is treated with a protease to cleave the peptide. In other embodiments, the peptide is phosphorylated. In yet other embodiments, the peptide is glycosylated. The peptide may be alpha-amidated. Other modifications may be performed on the peptide. The peptide may be modified chemically or enzymatically. Production ofExendin-4 in Pichia pastoris The production of recombinant exendin-4 in Pichia pastoris involves preparing the expression construct comprising a signal sequence, an optional propeptide or a tag which is followed by the 40 amino acid exendin-4 sequence in tandem. The signal sequence comprises the native exendin-4 signal sequence of 23 amino acid or the 19 amino acid alpha factor signal sequence. The propeptide comprises native exendin-4 propeptide of 24 amino acids or a 66 amino acid propeptide of alpha factor. A synthetic, Pichia codon optimized exendin-4 cDNA is engineered, and overlapping oligonucleotides of the sequence are prepared. The oligonucleotides are phosphorylated and annealed. PCR amplification is performed to recover the cDNA, which is cloned into the restriction sites of an appropriate vector carrying a selectable marker gene. The vector is selected from pPIC, pPIC3K, pPIC9K, or pHIL-D. These vectors include the AOX promoter to drive the expression of the exendin-4 gene. The vector carrying the synthetic exendin-4 cDNA is transformed into Pichia pastoris cells, and the transformed cells are plated on minimal medium. Transformed cells carrying the clone are then selected for. The transformed Pichia cells are plated on medium containing a selection agent, and positive clones are screened and/or selected. Fermentation with the positive clones are done, and the exendin-4 peptide is purified from Pichia cell-free supernatant using a combination of chromatographic techniques which include ion exchange, hydrophobic, gel filtration, and/or affinity chromatography. In certain embodiments, the exendin-4 peptide produced by the Pichia cells is a totally non-glycosylated product. Purification and Modification of Exendin-4 The recombinant peptide or peptide conjugate having the polypeptide sequence of exendin-4 is isolated and purified from the culture medium by separating the medium from the host cells, or if the peptide is produced in the cell, separating the cell debris after cell lysis. The peptide is then precipitate using salt or solvent and subjected to a variety of chromatographic procedures like ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and/or crystallization. The exendin-4 peptide has 40 amino acids. Optionally, the 39* amino acid, serine, is amidated. The 40 amino acid peptide is amidated in vitro to yield an exendin-4 peptide of 39 amino acids with the C-terminal amino acid amidated, that is, the C-terminal glycine is removed and the penultimate serine is amidated. In certain embodiments, the amidation is accomplished using a C-terminal alpha-amidating enzyme. The biological activity of exendin-4 and the 39 amino acid amidated version is determined by its ability to induce the secretion of insulin in a rat insulinoma cell line as described in more detail in the Examples below. Pharmaceutical Compositions Once the peptide have been prepared, it may be combined with a pharmaceutical excipient to form a pharmaceutical composition. As would be appreciated by one of skill in this art, the excipients may be chosen based on the route of administration as described below, the peptide, time course of delivery of the peptide, disease or condition being treated, etc. Pharmaceutical compositions of the present invention and for use in accordance with the present invention may include a pharmaceutically acceptable excipient or carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF), and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions of this invention can be administered to humans and/or to animals, orally, rectally, parenterally, intracistemally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), transdermally, subcutaneously, bucally, or as an oral or nasal spray. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Uses The insulinotropic peptides produced by the inventive system may be used in research activities or in the treatment of diseases or conditions. A therapeutically effective amount of the peptide may be delivered to an animal to treat any disease requiring the production of insulin. Such diseases include diabetes and hyperglycemia. The peptide may be used to treat other gastroinstestinal or endocrine diseases. For example, the peptide may be used to reduce gastric motility and/or delay gastric emptying. The peptide may also be administered to prevent hyperglycemia. In other embodiments, the peptide may be used to reduce food intake or to treat obesity. Exendin-4 is particularly useful in treating these diseases and conditions in humans and other animals. These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims. Examples Example l-Cloning of exendin-4 into Pichia pastoris expression vector The nucleotide sequence coding for exendin-4 was synthesized using overlapping oligonucleotides; ten oligonucleotides were synthesized to cover the exendin-4 gene in both directions. These oligonucleotides had a 14 base pair overlapping region with the next successive primer. These overlapping oligonucleotides were phospharylated, annealed, and ligated to each other to form the nucleotide sequence coding for exendin-4. The ligation mix was used as a template for amplification by the polymerase chain reaction (PCR) of the nucleotide sequence coding for exendin-4. PCR was performed according to standard protocols using Pfu turbo polymerase. After an initial denaturation step of 4 minutes at 94 X, the reaction mixture was subjected to 30 amplification cycles (denaturation at 94 ^'C for 30 sec; annealing at 60 ^'C for 30 sec; extension at 72 °C for 60 sec.) in a thermal cycler. The 140 bp PCR fragment obtained after amplification was cloned into the Xhol and EcoRl sites of the Pichia pastoris expression vector, pPIC9K (Invitrogen, San Diego, CA) to yield the recombinant expression vector. The nucleotide sequence coding for exendin-4 was cloned in frame with the Saccharomyces cerevisiae Mat alpha signal peptide present in the pPIC9K vector or its native exendin-4 signal peptide for secretion. The expression of the exendin-4 gene in the pPIC9K vector is under the control of the methanol-inducible alcohol oxidase (AOXl) promoter. Example 2-Expressioii of Exendiii-4 in Pichia pastoris host Transformation The plasmid having the Pichia codon optimized nucleotide insert was introduced into Pichia pastoris, GS115 strain {his4, Mut"^) by electroporation as described in the Invitrogen manual. The conditions used for electroporation were charging voltage of 1.5 kV, capacitance of 25 ^F, and resistance of 200 Q. The transformed cells were plated on minimal medium (YNBD) lacking histidine and incubated at SO'^C for 3 days. a^icreeningjor expression Single colonies of transformants were picked and grown in microtitre plates containing YPD (1% yeast extract, 2% Bacto-Peptone, 2% dextrose) at 30^0 overnight. The pre-grown cultures were replica-plated onto YPD agar plates with Genticin (2 mg/ml) and incubated at 30°C. Clones growing on YPD-Genticin (2 mg/ml) plate were selected for carrying out expression studies. Shake flask expression of transformed Pichia clones The selected clones, were inoculated into YNBG minimal medium (1.34% yeast nitrogen base without amino acids, 2% glycerol) and incubated for 16-18 hours at 30°C. The cultures were expanded in BMGY medium (1% yeast extract, 2% Bacto-Peptone, 1.34% YNB, 2% glycerol, and 100 mM phosphate buffer pH 6.0). After growing for 48 hrs at 30°C, the cells were pelleted and resuspended in BMMY (1% yeast extract, 2% Bacto-Peptone, 1.34% YNB, 0.5% methanol, and 100 mM phosphate buffer pH 6.0). Induction was carried out for 4 days at 30'C. The clones were checked for exendin-4 expression by analyzing the cell-free supernatant using SDS- PAGE followed by visualization with Coomassie blue staining. The amount of exendin-4 expression was determined by HPLC analysis and mass spectrometric analysis. The clone showing the highest level of exendin-4 secretion was selected. C-terminal a-amidation of exendin-4 using a-amidating enzyme The secreted form of exendin-4 has 40 amino acids. In vitro amidation of the C-terminus yields an exendin-4 peptide of 39 amino acids with the C-terminal amino acid serine amidated. The enzyme used is a C-terminal alpha amidating enzyme. The biological activity of exendin-4 (with 40 amino acids) and the 39 amino acid amidated version is determined by its ability to induce the secretion of insulin in a rat insulinoma cell line. The insulin secreted is detected by ELISA. The 40 amino acid peptide was converted to an amidated 39 amino acid version of exendin-4 with the loss of the C-terminal glycine and amidation of the penultimate serine. This requires the use of the C-terminal alpha amidating enzyme. The optimum pH of the enzymatic reaction is 5 to 6. The reaction was conducted at a temperature between 30 °C to 40 °C. The components in the reaction mixture included 5 to 10 mg/ml of exendin-4, 250 to 2000 units/ml of the amidating enzyme, 1 to 10 [iM/ml of catalase, 25 \|aM of copper sulfate, and 0.2 to 2 g/L of L-ascorbic acid. The reaction was completed in four hours with an overall yield of 60% of the amidated exendin-4. Example 3-Clonuig of codon optimized exendin-4 A codon optimized nucleotide sequence coding for exendin-4 was synthesized by polymerase chain reaction (PCR) using overlapping oligonucleotides. Ten oligonucleotides were synthesized to cover the exendin-4 gene along both strands having enough base pairs overlapping region with successive primers. The overlapping oligonucleotides were phosphorylated in a reaction mixture containing lOx PNK buffer, T4 polynucleotide kinase, and dATP, and the phosphorylated oligonucleotides were ligated using T4 DNA ligase. The ligation mixture was used as a template for amplification of the exendin-4 gene by PCR using the oligonucleotides, 5'-CTC GAG AAA AGA CAT GGA GAA GGA ACA TTT ACA TC-3'(SEQ ID NO: 6) (forward primer) and 5'-GAA TTC TTA TCC AGA TGG TGG-3' (SEQ ID NO: 7) (reverse primer). The PCR reaction was performed according to standard protocols, and amplification was done in a final volume of 50 jaL using Pfu Turbo polymerase. The amplification conditions included an initial denaturation of 4 minutes at 94 °C for one cycle, followed by 30 cycles (denaturation at 94 ®C for 30 seconds; annealing at 60 °C for 30 seconds; extension at 72 '^C for 60 seconds) and a final extension for 10 minutes for one cycle in a Perkin-Elmer Thermal cycler. Example 4 The 140 bp PCR fragment obtained after amplification was cloned into the vector pTZ57R using a TA cloning kit, in a reaction mix containing 3 \xl of vector, 2 ^1PEG4000 PCR buffer, and 1 ^l T4 DNA ligase. DH5a competent cells were transformed, and positive clones containing the exendin-4 gene fragment were screened by restriction digestion and sequencing. Example 5-Cloning of exendm-4 in Pichia pastoris The exendin-4 fragment in example 2, was excised using EcoRl and Xhol and sub cloned in frame into the Pichia pastoris expression vector pPIC9K vector for secretion. The expression of the exendin-4 gene was placed under the control of the methanol-inducible alcohol oxidase (AOX) promoter. The expression vector was transformed into Pichia pastoris by electroporation as described in the invitrogen manual. Single colonies of transformed cells were checked for G418 antibiotics resistance, and the selected transformants were used to carry out expression studies. Example 6-Cloning of exendiii-4 in Pichia pastoris using a spacer sequence The codon optimized nucleotide sequence coding for exendin-4 is cloned in tandem with and a spacer sequence coding for EAEA at its n-terminal end into a Pichia pastoris vector pPIC9K. The expression vector carrying the insert was transformed into Pichia pastoris. Upon expression of the polypeptide the Mat-a which ends with Lys-Arg is cleaved from the peptide by the action of Kex2 while the spacer peptide is cleaved by a Stel3 protease allowing the release of exendin-4. The expression level of the secreted exendin-4 in the medium was 300 mg/L. Example 7-Fermentation of Pichia pastoris for expression of exendin-4 The frozen (-70 °C) cells of Pichia sp. are cultivated in 250 ml flask containing 50 ml growth medium (1% yeast extract, 2% peptone, 10% IM phosphate buffer of pH 6.0, 0.67% yeast nitrogen base, and 0.1% glycerol) at 28-32 °C and 220-260 rpm. The seed cells are cultivated in 2 L fermenter containing one liter of fermentation medium consisting of 4% glycerol, 0.01% calcium sulfate, 2% potassium sulfate, 1.4% magnesium sulfate, and 0.4% potassium hydroxide. Fermentor experiments with the above medium were performed at different temperatures ranging from 20 'C to 30 ^C, pH 5.0, aeration rate 0.5-'2.0 wm. Agitation speed was increased gradually fi"om 350 to 1200 rpm to maintain the dissolved oxygen above 30%. Biomass was built up to 300 g/L by 50% glycerol feeding. Aseptically IL broth was harvested and 50 ml of broth was dispensed in each of twelve 250 ml flasks. All flasks were incubated at different temperatures ranging from 20 X to 30 °C at 220 to 260 rpm. 100 jiL of methanol was added every day, and after five days analysis for exendin-4 expression was performed. At 20 "^C, assay for exendin-4 after five days showed 0.0443 g/L. At 22 °C, assay after five days showed 0.0467 g/L. At 24 °C, assay after five days showed 0.0567 g/L. At 26 'C, assay after five days showed 0.0867 g/L. At 28 °C, assay after five days showed 0.0905 g/L. At 30 ^C, assay after five days showed 0.0685 g/L Example 8-Fermentation of recombinant Pichia for production of exendin-4 The frozen (-70 ^C) cells oiPichia sp are cultivated in a 250 ml flask containing 50 ml growth medium (1% yeast extract, 2% peptone, 10% IM phosphate buffer (pH 6.0), 0.67% yeast nitrogen base, and 0.1% glycerol) at 28-^32 °C and 220^-260 rpm. The seed cells are cultivated in a 2 L fermenter containing one liter of fermentation medium (3.5% glycerol, 0.01% calcium sulfate, 1% potassium sulfate, 1% magnesium sulfate, 0.25% potassium hydroxide). Fermenter experiments with the above medium were performed at different temperatures ranging from 20 € to 30 *€, pH 5.0, aeration rate 0.5'-2.0 wm. Agitation speed was increased gradually from 350 to 1200 rpm to maintain the dissolved oxygen above 30%. Biomass was built up to 285 g/L by 50% glycerol feeding, and then methanol feeding was carried out. Samples were taken every 24 hour after methanol feeding, and exendin-4 levels were determined using HPLC. At 20 °C, after five days exendin-4 assay showed 0.23 g/L. At 22 °C, after five days assay showed 0.37 g/L. At 24 ^'C, after five days assay showed 0.67 g/L. At 26 °C after five days assay showed 0.72 g/L. At 28 T, after five days assay showed 0.75 g/L. At 30 °C, assay showed 0.67 g/L. Example 9-Purification of exendin-4 Exendin-4 at a concentration of 300 mg/L was loaded onto a CM-Sepharose cation exchange column at pH 4, and the peptide was eluted at pH 5. The recovery was approximately 65%, and the purity was approximately 80%. In a subsequent reverse phase (C8) HPLC step, pore size 13 ^, using an acetonitrile-water gradient from 20 to 60%, the exendin-4 peptide was eluted with a purity of 95.5%. The recovery in this step was 20%. ESI-MS of the purified peptide showed the exact mass of exendin-4 which is calculated to be 4424.66, and N-terminal sequencing indicated the sequence to be HGEGTFTSDL (SEQ ID NO: 8), which is the known sequence of exendin-4. Example 10-Altemative purification of exendin-4 Exendin-4 at a concentration of 300 mg/L was loaded onto a SP-Sepharose cation exchange column at pH 3.8, and the peptide was eluted at pH 5.4. The recovery was approximately 61%, and the purity was approximately 80%. In a subsequent reverse phase (C8) HPLC step, pore size 13 /x, using acetonitrile-water gradient from 30 to 50%, the exendin-4 peptide was eluted with a purity of 97.5%. The recovery in this step was 20%. ESI-MS showed the exact mass of exendin-4 which is calculated to be 4424.66, and the N-terminal sequencing indicated the sequence to be HGEGTFTSDL (SEQ ID NO: 8), which is the known sequence of exendin-4. Example 11-Peptide mass fingerprinting using trypsin digestion After partial digestion using trypsin followed by analysis using ESI-MS, four observed masses as mentioned below correlated well to the sequences shown. After the data acquisition, data was converted to Mascot file and uploaded on MATRIXSCIENCE website for peptide mass finger printing for identification. The complete sequence of the expressed exendin-4 peptide could be unambiguously determined. Observed Mass Sequence correlated 1278.83 HGEGTFTSDLSK (SEQ ID NO: 9) 991.65 QMEEEAVR (SEQ ID NO: 10) 948.83 LFIEWLK (SEQ ID NO: 11) 1082.63 NGGPSSGAPPPSG (SEQ ID NO: 12) Example 12-C-termmal alpha amidation of exendin-4 The 40 amino acid peptide was converted to an amidated 39 amino acid exendin-4 with the loss of the C-terminal glycine and amidation of the penultimate serine. This step utilized a C-terminal alpha amidating enzyme. The optimum pH of the enzymatic reaction is 5.4. The reaction was conducted at a temperature of 35 °C. The components in the reaction mixture were 8 mg/ml of exendin-4, 750 units/ml of the amidating enzyme, 8 \|aM/ml of catalase, 25 \|iM of copper sulfate, and 2 g/L of L-ascorbic acid. The reaction was completed in four hours with an overall yield of 60% of the amidated exendin-4. Example 13-Alternative C-terminal alpha amidation of exendin-4 The 40 amino acid peptide was converted to an amidated 39 amino acid exendin-4 with the loss of the C-terminal glycine and amidation of the penultimate serine. This required the use of a C-terminal alpha amidating enzyme. The optimum pH of the enzymatic reaction is 6. The reaction was conducted at a temperature of 37 °C. The components in the reaction mixture included 10 mg/ml of exendin-4,1500 units/ml of the amidating enzyme, 10 \|iM/ml of catalase, 25 \|aM of copper sulfate, and 1.2 g/L of L-ascorbic acid. The reaction was completed in four hours with an overall yield of 63% of amidated exendin-4. Example 13-Biological assay of exendin-4 The rat insulinoma cell line Rinm5F was used for these studies. This cell line expresses the GLP-1 receptor to which exendin-4 binds and induces insulin secretion. The secreted insulin was measured by ELISA. The basal insulin secretion was around 3 to 5 pmol/mg whole cell protein. After treatment with exendin-4 for 1 hour, the insulin secretion by these cells increased from 18 to 28 pmol/mg whole cell protein in three independent experiments. Other Embodiments The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. Claims What is claimed is: 1. A method of producing insulinotropic peptide having biological activity, the method comprising steps of: providing a host cell transformed with a polynucleotide vector encoding the polypeptide with insulinotropic activity; and growing the host cell under suitable conditions to produce the polypeptide. 2. The method of claim 1, wherein the step of providing comprises transforming a host cell with a vector comprising a polynucleotide encoding the polypeptide with insulinotropic activity. 3. The method of claim 1 further comprising the step of isolating the polypeptide. 4. The method of claim 3, wherein the step of isolating the polypeptide comprises isolating the polypeptide from the medium the host cells are growing in, 6. The method of claim 3 further comprising alpha-amidating the polypeptide. 7. The method of claim 1, wherein the cells are yeast cells. 8. The method of claim 1, wherein the cells are Saccharomyces cerevisiae. 9. The method of claim 1, wherein the cells are Pichiapastoris. 10. The method of claim 1, wherein the polypeptide is selected from the group consisting of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and an exendin peptide. 11. The method of claim 1, wherein the polypeptide is exendin-4. 12. The method of claim 1, wherein the host cell is Pichiapastoris, and the polypeptide is exendin-4. 13. The method of claim 11 or 12, wherein the exendin-4 polypeptide is not glycosylated by the host cell. 14. The method of claim 11 or 12, wherein the exendin-4 polypeptide has the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2). 15. The method of claim 1, wherein the polynucleotide vector is a plasmid vector. 16. The method of claim 12, wherein the polynucleotide vector comprises a promoter selected from the group consisting of AOXl and A0X2. 17. The method of claim 1, wherein the step of growing the cells comprises growing the cells under conditions which select for the presence of the polynucleotide vector. 18. The method of claim 1, wherein the step of growing the host cells comprises growing the cells under conditions which induce the expression of the polypeptide. 19. The method of claim 18, wherein the step of growing the host cells comprises adding methanol to the host cells. 20. A polynucleotide encoding exendin-4 optimized for expression of exendin-4 in Pichiapastoris. Zl. ine polynucleotide of claim 20, wherein the amino acid sequence of exendin-4 comprises HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2). 22. The polynucleotide of claim 20, wherein the amino acid sequence of exendin-4 comprises HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 3). 23. The polynucleotide of claim 20, wherein the sequence comprises CTC GAG AAA AGA CAT GGA GAA GGA ACA TTT ACA TCT GAT TTG TCT AAA GAA ATG GAA GAA GAA GOT GTT AGA TTG TTT ATT GAA TGG TTG AAA AAC GGA GGA CCA TCT TCT GGA GCT CCA CCA CCA TCT GGA TAA GAA TTC (SEQ ID NO: 5). 24. The polynucleotide of claim 20 further comprising a sequence encoding a signal peptide. 25. The polynucleotide of claim 24, wherein the signal peptide is Mat alpha signal peptide or exendin-4 signal peptide. 26. The polynucleotide of claim 20 further comprising a sequence encoding a propeptide. 27. The polynucleotide of claim 26, wherein the propeptide is Mat alpha propeptide or exendin-4 propeptide. 28. The polynucleotide of claim 20 further comprising a sequence encoding a signal peptide and a propeptide. ^ 29. The polynucleotide of claim 20 further comprising a sequence encoding a purification tag. 30. A vector comprising a sequence encoding exendin-4 operable linked to a promoter. 31. The vector of claim 30, wherein the vector is a plasmid. 32. The vector of claim 30, wherein the vector is replicated in Pichia pastoris. 33. The vector of claim 30, wherein the vector provides for expression of the encoded exendin-4 peptide in Pichia pastoris. 34. The vector of claim 30, wherein the promoter is the AOX promoter. 35. The vector of claim 30, wherein the vector is derived from a vector selected from the group consisting of pPIC, pPIC3K, pPIC9K, and pHIL-D. 36. The vector of claim 30, further comprising a polynucleotide encoding a signal peptide. 37. A Pichia pastoris cell comprising a vector encoding exendin-4. 38. The cell of claim 40, wherein the vector is a vector of one of claim 30-37. 39. An exendin-4 polypeptide of the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 3), wherein the C-terminal serine residue is amidated. 40. The exendin-4 polypeptide of claim 39, wherein the polypeptide is not glycosylated. 41. A pharmaceutical composition comprising exendin-4 and a pharmaceutically acceptable excipient. 42. A pharmaceutical composition comprising exendin-4 prepared by the method of claim 1 and a pharmaceutically acceptable excipient. 43. A method of treating a patient with diabetes or preventing hyperglycemia, the method comprising steps of: administering to a patient a pharmaceutically acceptable amount of exendin-4. 44. A method of reducing food intake in a subject, the method comprising steps of: administering to a subject a pharmaceutically acceptable amount of exendin-4. 45. A method of alpha-amidating an exendin-4 polypeptide, the method comprising steps of: providing an exendin-4 polypeptide; and contacting the polypeptide with an C-terminal alpha-amidating enzyme under suitable conditions to yield an alpha-amidated exendin-4 polypeptide. 46. The method of claim 45, wherein the exendin-4 polypeptide has the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2); and wherein the alpha-amidated exendin-4 polypeptide has the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 3), wherein the C-terminal serine is amidated.

Full Text

Field of the Invention
The present invention provides a method of producing an insulinotropic peptide (e.g., exendin-4) by cloning and expressing the native peptide or an engineered form of the peptide in microorganisms such as yeast.
Background of the Invention
A number of hormones that lower blood glucose levels are released from the gastrointestinal mucosa in response to the presence and/or absorption of nutrients in the gut. These hormones include gastrin, secretin, glucose-dependent insulinotropic polypeptide (GIF), and glucagon-like peptide-1 (GLP-1).
GLP-1 is a proteolytic product of proglucagon, a 180 amino acid peptide which also gives rise to glucagon peptide (Orskov 1992 Diabetologia 35; 701-711; Drucker 1998, Diabetes Al\ 159-169; each of which is incorporated herein by reference). GLP-1 has a number of functions: (i) it is a physiological hormone that enhances the effect on insulin secretion in normal humans and is therefore an incretin hormone; (ii) GLP-1 also lowers glucagon concentrations, slows gastric emptying, stimulates (pro) insulin biosynthesis and enhances insulin sensitivity (Nauck 1997, Horm. Metab. Res, 47; 1253-1258; incorporated herein by reference); (iii) the peptide also enhances the ability of the beta-cells to sense and respond to glucose in subjects with impaired glucose tolerance (Byrne 1998, Eur, J. Clin. Invest. 28; 72-78; incorporated herein by reference); and (iv) the insulinotropic effect of GLP-1 in humans increases the rate of glucose disappearance partly because of increased insulin levels and partly because of enhanced insulin sensitivity (D'Alessio 1994, J. Clin. Invest, 93; 2263-2266; incorporated herein by reference).
However, a major pharmacological drawback to native GLP-1 is its short half-life. In humans, GLP-1 is rapidly degraded by dipeptidyl peptidase (DPP-IV) into GLP-1 (9-36) amide, which acts as an endogenous GLP-1 receptor antagonist (Deacon, 1998 Diabetologia 41; 271-278; incorporated herein by reference). Several strategies have been proposed to limit this problem, for example, using inhibitors of

DPP-IV, or using DPP-IV-resistant analogues of GLP-1 (Deacon et al, 1998, Diabetes 47; 764-769; Ritzel, 1998 J. Endocrinol. 159; 93-102; US Patent 5545618, Pederson, 1998 Diabetes 47; 1253-1258; each of which is incorporated herein by reference).
Exendins, another family of peptides that lower blood glucose levels have some sequence similarity (53%) to GLP-1 (Goke et al, 1993 J. Biol Chem. 268; 19650-19655; incorporated herein by reference). The exendins are found in the venom of the Gila Monster (Raufman 1996 Reg, Peptides, 61; 1-18; incorporated herein by reference) and the Mexican beaded lizard. Exendin-4 found in the venom of the Gila Monster {Heloderma suspectum) is of particular interest. Published PCT international patent application, WO 98/35033, which is incorporated herein by reference, discloses the cDNA encoding proexendin peptide, including exendin and other novel peptides, its isolation, and antibodies which specifically recognize such peptides (Pohl et al 1998, J. Biol Chem, liy, 9778-9784; incorporated herein by reference). Exendin-4 has been shown to be a strong GLP-1 agonist in isolated rat insulinoma cells. This is expected since the His-Ala domain of GLP-1 recognized by DPP-IV is not present in exendin-4, which has the sequence His-Gly instead (Goke et. al, 1993, J. Biol Chem. 268; 19650-19655; incorporated herein by reference).
Sequences of GLP-1 and Exendin-4
GLP-l (7-37) HAEGTFTSDVSSYLEGQAAKEFLWVLVKGRG (SEQ ID NO:
1)
EXENDIN-4 (1-40) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG
(SEQ ID NO: 2)
Exendin-4 given systematically lowers blood glucose levels by 40% in diabetic db/db mice (WO 99/07404; incorporated herein by reference). Recently, Grieg et al (Diabetologia 1999 42; 45-50; incorporated herein by reference) has shown a long lasting blood glucose lowering effect of once daily intraperitoneal injections of exendin-4 in diabetic ob/ob mice. U.S. Patent 5,424,286 by Eng, which is

incorporated herein by reference, discloses that a considerable portion of the N-terminal sequence is essential in order to preserve insulinotropic activity whereas an N-terminally truncated exendin (exendin-4 (9-39)) has inhibitory properties.
The use of exendin-4 and exendin agonists has been proposed for the treatment of diabetes mellitus, reducing gastric motility and delaying gastric emptying, and preventing hyperglycemia (U.S. Patent 5,424,286; WO 98/05351; each of which is incorporated herein by reference) as well as for reducing food intake (WO 98/30231; incorporated herein by reference).
However, the number of amino acid residues in a peptide sequence has a dramatic influence on the production costs of peptides made by solid phase synthetic chemistry. The cost of manufacturing a 50-mer peptide is at least 5 times greater than the cost of a 10-mer peptide. Solid phase peptide synthesis also requires the use of corrosive solvents such as TFS and hydrofluoric acids, a number of synthetic steps required to produce the peptide, and subsequent purification of the peptide. Therefore, there is a need for efficiently and cost effectively producing large quantities of biologically active exendin-4 and other insulinotropic peptides for the treatment of these conditions.
Summary of the Invention
The present invention provides a system for preparing insulinotropic peptides by heterologous gene expression in microorganisms. This system provides for large-scale recombinant production of peptides such as exendin-4. The system provides a way of producing large quantities of biologically active peptides without resorting to solid phase peptide synthesis. The peptides produced using the inventive system may be used in formulating pharmaceutical compositions. The peptides and compositions thereof may be used in treating diabetes mellitus, reducing gastric motility, delaying gastric emptying, preventing hyperglycemia, and reducing appetite and food intake.
The present invention provides a method of producing insulinotropic peptide having biological activity, the method comprising steps of:

- providing a host cell transformed with a polynucleotide vector encoding the polypeptide with insulinotropic activity; and
- growing the host cell under suitable conditions to produce the polypeptide.
In producing an insulinotropic peptide by heterologous gene expression in a microorganism, the encoding polynucleotide sequence for the peptide, preferably as part of a vector, is transformed into a host cell of the microorganism. The expression of the gene encoding the insulinotropic peptide is controlled by a promoter (e.g., an inducible promoter or constitutive promoter). Preferably, the promoter is a strong promoter, more preferably a strong inducible promoter, which allows for the production of large quantities of the insulinotropic peptide. The cells transformed with the gene may be fungal (e.^., yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris)). For expression in P, pastoris, the promoter used to drive the expression of the gene is preferably the AUGl promoter, the GAP promoter (a strong constitutive promoter), or the AOXl or A0X2 promoter (a strong inducible promoter); and the vector may be derived from a known or commercially available vector such as pPIC, pPIC3K, pPIC9K, or pHIL-D. For expression in S. cerevisiae, the promoter used may be the CUPI promoter, ADH promoter, the TEFl promoter, or the GAL promoter. The polynucleotide sequence encoding the insulinotropic peptide being expressed may be optimized for expression in the particular organism {e.g., codon usage may be optimized for use in the host microorganism). The polynucleotide sequence may optionally include leader sequences, signal sequences, pre-/pro-peptide sequences, tags for processing, tags for detection or purification, fusion peptides, etc. In certain embodiments, the gene encodes a signal peptide. The signal peptide may direct the processing or secretion of the peptide. In certain embodiments, the signal peptide is the signal peptide of exendin-4 or the signal peptide of mat alpha factor. In certain embodiments, the gene encodes a propeptide, and the propeptide includes the native exendin-4 propeptide or the propeptide of mat alpha factor (see WO 98/35033; Pohl et al,J. Biol Chem. 273:9778-9784, 1998; each of which is incorporated herein by reference).

The transformed host cells are selected for the presence of the vector (e.g., antibiotic resistance, ability to grown on media which does not contain a particular nutrient) and/or expression of the exogenous gene encoding the insulinotropic peptide. The selected cells are then grown under conditions suitable for expression of the gene encoding the peptide. In certain embodiments, the medium in which the cells are grown include an agent, such as methanol, which induces the expression of the gene encoding the peptide. In other embodiments, when a constitutive promoter is used, an agent for inducing the expression for the exogenous gene is not necessary. The transformed host cell produces the peptide. Preferably, peptide is properly processed by the host cell. That is, the peptide is properly folded and post-translationally modified. In certain embodiments, the peptide is secreted it into the growth medium. The recombinantly produced peptide is then isolated and purified from the host cells or from the medium, if the peptide has been secreted. The purified peptide may be further modified chemically or enzymatically (e.g-., alpha amidation, cleavage).
The isolated, modified, and/or purified peptide may be used in formulating pharmaceutical compositions and treating patients with a particular disease or condition involving glucose metabolism/uptake. The compositions may include insulinotropic peptides of a specific activity greater than the activities seen in peptides produced by other means such as solid phase peptide synthesis. These compositions may be used to treat such endocrine diseases as diabetes mellitus. These compositions may also be used to treat conditions such as reduced gastric motility, delayed gastric emptying, hyperglycemia, and obesity.
In one aspect, the insulinotropic peptide, exendin-4, is produced. The peptide may be produced in any microorganism, preferably Pichia pastoris. The production of recombinant exendin-4 in Pichia pastoris involves preparing the expression construct comprising a signal sequence, a pro-peptide, a pre-peptide, and/or a tag, which is followed by the 40 amino acid exendin-4 sequence in tandem. In certain embodiments, the signal sequence comprises the native exendin-4 signal sequence of 23 amino acid or a 19 amino acid alpha factor signal sequence. The propeptide comprises the native exendin-4 propeptide of 24 amino acids or a 66 amino acid

propeptide of alpha factor. The tag may aid in the processing of the peptide (Kjeldsen et ah Biotechnol Appl Biochem. 29:79-86, 1999; incorporated herein by reference). In certain embodiments, the tag is a charged ammo acid sequence such as the EAEA spacer described in Example 6. A Pichia codon optimized nucleotide sequence coding for exendin-4 optionally with a signal peptide or propeptide is prepared. The synthetic nucleotide sequence is then cloned into an appropriate vector carrying a selectable marker gene, such as pPIC, pPIC3K, pPIC9K, or pHIL-D. The vector is transformed into Pichia pastoris cells, and transformed cefls carrying the vector are selected. The selected clone is fermented on a large enough scale to produce the desired quantity of exendin-4 peptide. The exendin-4 peptide so produced into the broth need no further processing steps to make it a fully functional peptide unlike that produced in bacterial system (EP 978565 Ohsuye et al Suntory limited; incorporated herein by reference). The peptide is purified from the medium using protein purification techniques known in the art. The exendin-4 peptide is preferably not glycosylated by the Pichia host cells. Lack of glycosylation is also an advantage because the Exendin-4 is fully functional and it leads to a simpler and easier purification process and results in improved yields of the peptide. The purified exendin-4 peptide may be further chemically or enzymatically modified (e,g,, C-terminal alpha amidation). The exendin-4 peptide may be used in pharmaceutical compositions for administration to a subject (e.g., humans).
The invention also provides kits and reagents useful in recombinantly producing insulinotropic peptides in microorganisms. These kits may include polynucleotides, nucleotides, vectors, enzymes, cells, buffers, media, vials, plates, Eppendorf tubes, chromatographic materials, instructions, etc. Preferably, the kit is conveniently packaged for use in a laboratory setting. In certain embodiments, Pichia pastoris cells transformed with a vector encoding exendin-4 are included in the kit. In certain embodiments, the kit may also include the materials necessary for purifying the overexpressed peptide.

Definitions
"Animal": The term animal, as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). An animal may be a transgenic animal.
"Effective amount": In general, the "effective amount" of an active agent or the microparticles refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a peptide or pharmaceutical composition may vary depending on such factors as the desired biological endpoint, the peptide to be delivered, the disease or condition being treated, etc,
"Peptide" or "protein": According to the present invention, a "peptide" or "protein" comprises a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids {i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification {e.g., alpha amindation), etc. In a preferred embodiment, the modifications of the peptide lead to a more stable peptide {e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide. In certain embodiments, the modifications of the peptide lead to a more biologically active peptide.
"Transformation": The term "transformation" as used herein refers to introducing a vector comprising a nucleic acid sequence into a host cell. The vector

may mtegrate itselt or a portion or itseii mro me cnromosome oi me ceiis, or me vector may exist as a self-replicating extrachromosomal vector. Integration is considered in some embodiments to be advantageous since the nucleic acid is more likely to be stably maintained in the host cell. In other embodiments, an extrachromosomal vector is desired because each host cell can contain multiple copies of the vector.
Brief Description of the Drawing
Figure 7 is a schematic showing modified versions of the exendin-4 gene including signal peptide and propeptide sequences. These modified versions of exendin-4 may be used in the Pichia pastoris expression system.
Detailed Description of Certain Preferred Embodiments of ttie Invention
The inventive system for recombinantly producing insulinotropic peptides is particularly usefiil in preparing large quantities of biologically active peptide for use in pharmaceutical compositions or for research purposes. The invention provides methods of preparing the polynucleotide and recombinant cells for producing the peptide as well as methods of recombinantly producing the peptide itself The invention also includes polynucleotide sequences, vectors, cells, and kits usefiil in practicing the inventive methods, and pharmaceutical compositions and methods of using the recombinantly produced peptides.
As described above, recombinantly expressing an insulinotropic peptide in a host microorganism involves preparing a polynucleotide with the peptide-encoding sequence, introducing this sequence into a vector, transforming cells of the microorganism with the vector, and selecting for cells with the vector. These selected cells are then grown under suitable conditions to produce the insulinotropic peptide, which is purified from the cells or from the medium in which the cells were growing. The purified peptide is then used in pharmaceutical compositions to treat such diseases as diabetes or obesity.

Polynucleotide
The production of a recombinant protein/peptide begins with the preparation of a polynucleotide sequence that encodes the insulinotropic peptide, or a modified version of the peptide, to be produced. Preferably, the peptide has been characterized sufficiently that the primary sequence of the peptide is known. The sequence may include sections of the peptide that are cleaved from the final product such as signal sequences, tags, fusion peptides or proteins, and/or pre-/pro-sequences. In certain instances, the gene or cDNA encoding the peptide is known in the art or has been cloned so that the primary sequence of the peptide has been deduced from the nucleotide sequence. In other embodiments, the primary sequence of the peptide has been determined {e.g., by Edman degradation), but the gene or cDNA encoding the peptide has not been cloned. The sequence of the gene, cDNA, and/or peptide may be available in the scientific literature or in a genetic database such as GenBank. The present invention focuses on the production of insulinotropic peptides such as glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1 (GLP-1), and exendin family members (e.g., exendin-4); therefore, the sequences of these peptides, as well as variants, homologs, mutants, allelic variants, isotypes, polymorphisms, etc., thereof, are particularly useful. In certain embodiments, exendin-4 is expressed. Exendin-4 has the primary amino acid sequence
(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2)). The amidated form of exendin-4 with the C-terminal glycine residue removed may also be produced and has the sequence
((HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2 (SEQ ID NO: 3)). In certain embodiments, the exendin-4 peptide or its amidated form is not glycosylated. The pro-exendin gene product has the primary amino acid sequence (MKIILWLCVFGLFLATLFPISWOMPVESGLSSEDSASSESFASKIKRHGEGTFT SDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 4)). The underlined residues indicate the pro-sequence.
In designing the peptide sequence for expression in a recombinant microorganism, the peptide sequence may be altered from the wild type sequence.

The alteration may be a mutation of one or more of the amino acids found in the peptide. The alteration may be a deletion or addition of an amino acid or plurality of amino acids. In certain embodiments, a sequence of amino acids is added to the peptide for recombinant production. Such a sequence can be added at the ends or in the middle of the peptide; however, preferably, the sequence is added at the C-terminus or N-terminus of the peptide. For example, a signal sequence may be added, preferably at the N-terminus of the peptide. A propeptide sequence may be added. A prepeptide sequence may be added. The peptide may be fused with another protein or peptide. A cleavage site may also be added. In certain embodiments, the resulting peptide sequence contains at least a contiguous 10,15, 20, 25, 30, or 35 amino acid sequence at least 75%, 80%, 90%, 95%, 98%, or 100% homologous to the wild type sequence. In certain embodiments, there is at least a 20 amino acid sequence that is at least 95% homologous to the wild type sequence.
Once the peptide sequence has been designed, the corresponding polynucleotide which encodes the engineered peptide sequence can be prepared. As will be appreciated by one of skill in this art, due to the redundancy in the genetic code, more than one polynucleotide sequence can encode a peptide. Any polynucleotide sequence that encodes the engineered peptide can be used in the recombinant production of the peptide. However, in certain embodiments, the polynucleotide sequence is optimized for expression in a particular organism. That is, the codon(s) commonly used by the organism to encode a particular amino acid are used in generating the encoding polynucleotide sequence. For example, if one were producing a peptide in Pichiapastoris, it would be preferable to use the codons in the genetic code more commonly used by this organism in expressing its own proteins (see Table 1 below, and U.S. Patent 5,827,684, incorporated herein by reference). For expression in S. cerevisiae, the codon usage table for S. cerevisiae may be used (see ftp://genome-ftp.stanford.edu/pub/codon/ysc.gene.cod; incorporated herein by reference). In certain embodiments, the most often used codon for an amino acid is not used but instead the second or third most frequently used codon is used in constructing the polynucleotide sequence. As would be appreciated by one of skill in

this art, codon usage depends upon the organism being used to express the peptide. Therefore, a polynucleotide sequence used to express a protein in one organism may not be optimal for expression in a different organism.

In a particularly preferred embodiment, the invention provides a polynucleotide sequence encoding exendin-4, which has been optimized for expression in Pichia pastoris. The sequence is as follows: CTC GAG AAA AGA CAT GGA GAA GGA ACA TTT ACA TCT GAT TTG TCT AAA CAA ATG GAA GAA GAA GCT GTT AGA TTG TTT ATT GAA TGG TTG AAA AAC GGA GGA CCA TCT TCT GGA GCT CCA CCA CCA TCT GGA TAA GAA TTC (SEQ ID NO: 5). In certain embodiments, the polynucleotide sequence is at least 90%, 95%, 97%, 99%, or 100% homologous to the optimized sequence over the entire sequence. In other embodiments, at least a 25, 50, 75, or 100 nucleotide sequence is at least 90%, 95%, 97%, 99%, or 100% homologous to the analogous section of the optimized sequence. As would be appreciated by one of skill in this art a polynucleotide encoding a signal sequence, propeptide sequence, prepeptide sequence, or tag may be added to the sequence. In certain embodiments, the polynucleotide encoding the signal sequence, propeptide sequence, prepeptide sequence, or tag is provided by the vector the sequence is subcloned into. In such a case, the peptide encoding DNA sequence is inserted in frame into the open reading frame of the vector.
In other embodiments, the exendin-4 encoding polynucleotide is optimized for expression in S. cerevisiae. These sequence may also include signal sequences or prepeptide sequences.
Vector
In order to express a polynucleotide in a cell, the polynucleotide is typically incorporated into a vector. The vector may be prepared using any techniques known

in the art (please see, Molecular Cloning: A Laboratory Manual^ 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); the treatise, Methods in Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Ausubel et al Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); each of which is incorporated herein by reference). The vector may be a plasmid, cosmid, chromosome, artificial chromosome, virus, phase, naked piece of DNA, etc. In certain embodiments, the vector is a plasmid. Preferably, the plasmid can be amplified in E. coti. The vector may be DNA or RNA based. The vector may be double-stranded DNA or RNA, or single-stranded DNA or RNA. The vector may include other sequences, for example, selection markers (e.g., antibiotic resistance genes, biosynthetic genes (e.g., URA3, LEU2, TRPl, HIS3)), origins of replication (e.g., 2|im, ARS (autonomously replicating sequence), ori), housekeeping genes, restriction sites, etc. In certain embodiments, the vector includes a cloning site (i.e., polylinker) for inserting the gene to be expressed.
The cloning site may be flanked by a promoter, enhancer sequences, ribosomal binding site, transcription start site, TATA box, termination site, etc. In certain embodiments, a constitutive promoter is used to express the peptide-encoding gene. In other embodiments, an inducible promoter is used to express the peptide-encoding gene. Typically the promoter is species specific. Examples of promoters for use in P. pastoris include the AOXl, A0X2, AUGl, and GAP promoters. In certain embodiments, the AOXl promoter is used for expression in P. pastoris. Example of promoters useful in S. cerevisiae include the CUPl, ADH, TEFl, and GAL promoters. The vector is typically designed for use in a particular organism. In other embodiments, the vector is designed for use in Saccharomyces cerevisiae. In yet other embodiments, the vector is designed for use in Pichia pastoris. Examples of plasmid vectors for use in Pichia pastoris include pPIC, pPIC3K, pPIC9K, and pHIL-

D. In certain embodiments, the vector is commercially available.
In certain embodiments, the plasmid vector include a polynucleotide encoding exendin-4 for expression in P. pastoris. Preferably, the vector is a plasmid derived from pPIC, pPIC3K, pPIC9K, and pHIL-D.
Cells
The vector encoding the peptide to be expressed is used to transform a cell. The cell may be a fungal cell, in particular a yeast cell. In certain embodiments, the cell is a S. cerevisiae cell. In certain other embodiments, the cell is a Pichia pastoris cell. The cells may be obtained commercially or may be obtained from depositories such as the ATCC. In certain embodiments, the cells are wild type cells. In other embodiments, the cells have been engineered. In other embodiments, the cells have been altered by the hand of man.
The vector is used to transform the cell by any means known in the art (please see. Molecular Cloning: A Laboratory Manual^ 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); the treatise. Methods in Enzymology (Academic Press, Inc., N.Y.); Ausubel et al. Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); each of which is incorporated herein by reference). Techniques for getting the vector into the cell include electroporation, calcium phosphate precipitation, transformation of competent cells, viral infection, phage infection, etc. In certain embodiments, electroporation is used to get the vector into the cell.
After the gene encoding the peptide to be expressed is transformed into the cells, the cells with the gene may be selected. This selection may be done by growing the cells in a particular medium which requires the expression of a gene on the vector. For example, the vector may encode an antibiotic resistance gene and the medium may contain the antibiotic. Cells which not have the vector will be killed by the antibiotic whereas cells with the antibiotic resistance gene will be able to grow. In certain embodiments, the cells are grown in media lacking an essential nutrient. The

vector confers the ability to grow on such media; therefore, cells with the vector grow while cells without the vector cannot divide. In certain embodiments, expression of the peptide may be determined directly by immunoassay or enzymatic assay.
After cells containing the vector with the gene to be expressed are selected, they may be grown in medium under suitable conditions to produce the desired peptide. The medium used to grow the cells, aeration, temperature, and the time course for growing the cells and inducing the cells to produce the peptide is determined by one of skill in this art or can be determined empirically. In growing recombinant ftingi (e.g., P. pastoris), the cells are grown between 22°C-32'C, preferably between 26°C-30'C. The aeration of the fermentation culture maintains dissolved oxygen above 20%, 25%, or 30%. Preferably, dissolved oxygen is maintained above 30%. In certain embodiments, an agent may be added to the growth media to induce the expression of the gene. In other embodiments, the gene is expressed constitutively without the need for such an inducing agent. The peptide may be harvested from the culture after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10 days, or 14 days. In certain embodiments, the expression level of the insulinotropic peptide exceeds 100 mg/L. In other embodiments, the expression level exceeds 200 mg/L. In yet other embodiments, the expression level exceeds 300 mg/L.
The organism chosen for expression of the peptide should correctly fold and modify the peptide so that the peptide has the same or greater biological activity than the peptide derived from its natural source. The peptide may be phosphorylated, glycosylated, cleaved, etc. by the machinery of the host cell. In certain embodiments, the sites for glycosylation on the peptide are not glycosylated by the host cells. Preferably, the peptide is not glycosylated at any sites. The lack of glycosylation may lead to easier purification and/or improved yields of the peptide. In certain embodiments, signal peptides, pro-peptide sequences, and pre-peptide sequences are cleaved from the peptide. In other embodiments, the peptide is processed in the cell and then secreted into the media the cells are growing in.
After the peptide has been harvested from the fermentation, the peptide is

purified. The peptide may be purified from the cells, or if the peptide is secreted, the peptide may be purified from the media. If the peptide is purified from cells, the cells are lysed, and the peptide is purified from the cell lysate. The peptide may be purified using any methods known in the art including ammonium sulfate precipitation, column chromatography (e.g. ion exchange chromatography, size exclusion chromatography, affinity chromatography), FPLC, HPLC {e,g., reverse phase, normal phase), e^c. The peptide is purified to the desired level of purity. In certain embodiments, the final peptide is greater than 90% pure, 95% pure, 98% pure, or 99% pure, preferably greater than 98% pure.
The purified peptide may be used as is, or the peptide may be further modified. In certain embodiments, the peptide is treated with a protease to cleave the peptide. In other embodiments, the peptide is phosphorylated. In yet other embodiments, the peptide is glycosylated. The peptide may be alpha-amidated. Other modifications may be performed on the peptide. The peptide may be modified chemically or enzymatically.
Production ofExendin-4 in Pichia pastoris
The production of recombinant exendin-4 in Pichia pastoris involves preparing the expression construct comprising a signal sequence, an optional propeptide or a tag which is followed by the 40 amino acid exendin-4 sequence in tandem. The signal sequence comprises the native exendin-4 signal sequence of 23 amino acid or the 19 amino acid alpha factor signal sequence. The propeptide comprises native exendin-4 propeptide of 24 amino acids or a 66 amino acid propeptide of alpha factor.
A synthetic, Pichia codon optimized exendin-4 cDNA is engineered, and overlapping oligonucleotides of the sequence are prepared. The oligonucleotides are phosphorylated and annealed. PCR amplification is performed to recover the cDNA, which is cloned into the restriction sites of an appropriate vector carrying a selectable marker gene. The vector is selected from pPIC, pPIC3K, pPIC9K, or pHIL-D. These vectors include the AOX promoter to drive the expression of the exendin-4 gene. The vector carrying the synthetic exendin-4 cDNA is transformed into Pichia pastoris

cells, and the transformed cells are plated on minimal medium. Transformed cells carrying the clone are then selected for.
The transformed Pichia cells are plated on medium containing a selection agent, and positive clones are screened and/or selected. Fermentation with the positive clones are done, and the exendin-4 peptide is purified from Pichia cell-free supernatant using a combination of chromatographic techniques which include ion exchange, hydrophobic, gel filtration, and/or affinity chromatography. In certain embodiments, the exendin-4 peptide produced by the Pichia cells is a totally non-glycosylated product.
Purification and Modification of Exendin-4
The recombinant peptide or peptide conjugate having the polypeptide sequence of exendin-4 is isolated and purified from the culture medium by separating the medium from the host cells, or if the peptide is produced in the cell, separating the cell debris after cell lysis. The peptide is then precipitate using salt or solvent and subjected to a variety of chromatographic procedures like ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and/or crystallization.
The exendin-4 peptide has 40 amino acids. Optionally, the 39* amino acid, serine, is amidated. The 40 amino acid peptide is amidated in vitro to yield an exendin-4 peptide of 39 amino acids with the C-terminal amino acid amidated, that is, the C-terminal glycine is removed and the penultimate serine is amidated. In certain embodiments, the amidation is accomplished using a C-terminal alpha-amidating enzyme.
The biological activity of exendin-4 and the 39 amino acid amidated version is determined by its ability to induce the secretion of insulin in a rat insulinoma cell line as described in more detail in the Examples below.
Pharmaceutical Compositions

Once the peptide have been prepared, it may be combined with a pharmaceutical excipient to form a pharmaceutical composition. As would be appreciated by one of skill in this art, the excipients may be chosen based on the route of administration as described below, the peptide, time course of delivery of the peptide, disease or condition being treated, etc.
Pharmaceutical compositions of the present invention and for use in accordance with the present invention may include a pharmaceutically acceptable excipient or carrier. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF), and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions of this invention can be administered to humans and/or to animals, orally, rectally, parenterally, intracistemally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), transdermally, subcutaneously, bucally, or as an oral or nasal spray.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also

be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Uses
The insulinotropic peptides produced by the inventive system may be used in research activities or in the treatment of diseases or conditions. A therapeutically effective amount of the peptide may be delivered to an animal to treat any disease requiring the production of insulin. Such diseases include diabetes and hyperglycemia. The peptide may be used to treat other gastroinstestinal or endocrine diseases. For example, the peptide may be used to reduce gastric motility and/or delay gastric emptying. The peptide may also be administered to prevent hyperglycemia. In other embodiments, the peptide may be used to reduce food intake or to treat obesity. Exendin-4 is particularly useful in treating these diseases and conditions in humans and other animals.
These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

Examples Example l-Cloning of exendin-4 into Pichia pastoris expression vector
The nucleotide sequence coding for exendin-4 was synthesized using overlapping oligonucleotides; ten oligonucleotides were synthesized to cover the exendin-4 gene in both directions. These oligonucleotides had a 14 base pair overlapping region with the next successive primer. These overlapping oligonucleotides were phospharylated, annealed, and ligated to each other to form the nucleotide sequence coding for exendin-4. The ligation mix was used as a template for amplification by the polymerase chain reaction (PCR) of the nucleotide sequence coding for exendin-4.
PCR was performed according to standard protocols using Pfu turbo polymerase. After an initial denaturation step of 4 minutes at 94 X, the reaction mixture was subjected to 30 amplification cycles (denaturation at 94 ^'C for 30 sec; annealing at 60 ^'C for 30 sec; extension at 72 °C for 60 sec.) in a thermal cycler. The 140 bp PCR fragment obtained after amplification was cloned into the Xhol and EcoRl sites of the Pichia pastoris expression vector, pPIC9K (Invitrogen, San Diego, CA) to yield the recombinant expression vector. The nucleotide sequence coding for exendin-4 was cloned in frame with the Saccharomyces cerevisiae Mat alpha signal peptide present in the pPIC9K vector or its native exendin-4 signal peptide for secretion. The expression of the exendin-4 gene in the pPIC9K vector is under the control of the methanol-inducible alcohol oxidase (AOXl) promoter.
Example 2-Expressioii of Exendiii-4 in Pichia pastoris host
Transformation
The plasmid having the Pichia codon optimized nucleotide insert was introduced into Pichia pastoris, GS115 strain {his4, Mut"^) by electroporation as described in the Invitrogen manual. The conditions used for electroporation were charging voltage of 1.5 kV, capacitance of 25 ^F, and resistance of 200 Q. The transformed cells were plated on minimal medium (YNBD) lacking histidine and incubated at SO'^C for 3 days.

a^icreeningjor expression
Single colonies of transformants were picked and grown in microtitre plates containing YPD (1% yeast extract, 2% Bacto-Peptone, 2% dextrose) at 30^*0 overnight. The pre-grown cultures were replica-plated onto YPD agar plates with Genticin (2 mg/ml) and incubated at 30°C. Clones growing on YPD-Genticin (2 mg/ml) plate were selected for carrying out expression studies.
Shake flask expression of transformed Pichia clones
The selected clones, were inoculated into YNBG minimal medium (1.34% yeast nitrogen base without amino acids, 2% glycerol) and incubated for 16-18 hours at 30°C. The cultures were expanded in BMGY medium (1% yeast extract, 2% Bacto-Peptone, 1.34% YNB, 2% glycerol, and 100 mM phosphate buffer pH 6.0). After growing for 48 hrs at 30°C, the cells were pelleted and resuspended in BMMY (1% yeast extract, 2% Bacto-Peptone, 1.34% YNB, 0.5% methanol, and 100 mM phosphate buffer pH 6.0). Induction was carried out for 4 days at 30*'C. The clones were checked for exendin-4 expression by analyzing the cell-free supernatant using SDS- PAGE followed by visualization with Coomassie blue staining. The amount of exendin-4 expression was determined by HPLC analysis and mass spectrometric analysis. The clone showing the highest level of exendin-4 secretion was selected.
C-terminal a-amidation of exendin-4 using a-amidating enzyme
The secreted form of exendin-4 has 40 amino acids. In vitro amidation of the C-terminus yields an exendin-4 peptide of 39 amino acids with the C-terminal amino acid serine amidated. The enzyme used is a C-terminal alpha amidating enzyme.
The biological activity of exendin-4 (with 40 amino acids) and the 39 amino acid amidated version is determined by its ability to induce the secretion of insulin in a rat insulinoma cell line. The insulin secreted is detected by ELISA. The 40 amino acid peptide was converted to an amidated 39 amino acid version of exendin-4 with the loss of the C-terminal glycine and amidation of the penultimate serine. This

requires the use of the C-terminal alpha amidating enzyme.
The optimum pH of the enzymatic reaction is 5 to 6. The reaction was conducted at a temperature between 30 °C to 40 °C. The components in the reaction mixture included 5 to 10 mg/ml of exendin-4, 250 to 2000 units/ml of the amidating enzyme, 1 to 10 [iM/ml of catalase, 25 |aM of copper sulfate, and 0.2 to 2 g/L of L-ascorbic acid. The reaction was completed in four hours with an overall yield of 60% of the amidated exendin-4.
Example 3-Clonuig of codon optimized exendin-4
A codon optimized nucleotide sequence coding for exendin-4 was synthesized by polymerase chain reaction (PCR) using overlapping oligonucleotides. Ten oligonucleotides were synthesized to cover the exendin-4 gene along both strands having enough base pairs overlapping region with successive primers. The overlapping oligonucleotides were phosphorylated in a reaction mixture containing lOx PNK buffer, T4 polynucleotide kinase, and dATP, and the phosphorylated oligonucleotides were ligated using T4 DNA ligase.
The ligation mixture was used as a template for amplification of the exendin-4 gene by PCR using the oligonucleotides, 5'-CTC GAG AAA AGA CAT GGA GAA GGA ACA TTT ACA TC-3'(SEQ ID NO: 6) (forward primer) and 5'-GAA TTC TTA TCC AGA TGG TGG-3' (SEQ ID NO: 7) (reverse primer). The PCR reaction was performed according to standard protocols, and amplification was done in a final volume of 50 jaL using Pfu Turbo polymerase. The amplification conditions included an initial denaturation of 4 minutes at 94 °C for one cycle, followed by 30 cycles (denaturation at 94 ®C for 30 seconds; annealing at 60 °C for 30 seconds; extension at 72 '^C for 60 seconds) and a final extension for 10 minutes for one cycle in a Perkin-Elmer Thermal cycler.
Example 4
The 140 bp PCR fragment obtained after amplification was cloned into the vector pTZ57R using a TA cloning kit, in a reaction mix containing 3 \xl of vector, 2

^1PEG4000 PCR buffer, and 1 ^l T4 DNA ligase. DH5a competent cells were transformed, and positive clones containing the exendin-4 gene fragment were screened by restriction digestion and sequencing.
Example 5-Cloning of exendm-4 in Pichia pastoris
The exendin-4 fragment in example 2, was excised using EcoRl and Xhol and sub cloned in frame into the Pichia pastoris expression vector pPIC9K vector for secretion. The expression of the exendin-4 gene was placed under the control of the methanol-inducible alcohol oxidase (AOX) promoter. The expression vector was transformed into Pichia pastoris by electroporation as described in the invitrogen manual. Single colonies of transformed cells were checked for G418 antibiotics resistance, and the selected transformants were used to carry out expression studies.
Example 6-Cloning of exendiii-4 in Pichia pastoris using a spacer sequence
The codon optimized nucleotide sequence coding for exendin-4 is cloned in tandem with and a spacer sequence coding for EAEA at its n-terminal end into a Pichia pastoris vector pPIC9K. The expression vector carrying the insert was transformed into Pichia pastoris. Upon expression of the polypeptide the Mat-a which ends with Lys-Arg is cleaved from the peptide by the action of Kex2 while the spacer peptide is cleaved by a Stel3 protease allowing the release of exendin-4. The expression level of the secreted exendin-4 in the medium was 300 mg/L.
Example 7-Fermentation of Pichia pastoris for expression of exendin-4
The frozen (-70 °C) cells of Pichia sp. are cultivated in 250 ml flask containing 50 ml growth medium (1% yeast extract, 2% peptone, 10% IM phosphate buffer of pH 6.0, 0.67% yeast nitrogen base, and 0.1% glycerol) at 28-32 °C and 220-260 rpm. The seed cells are cultivated in 2 L fermenter containing one liter of fermentation medium consisting of 4% glycerol, 0.01% calcium sulfate, 2% potassium sulfate, 1.4% magnesium sulfate, and 0.4% potassium hydroxide. Fermentor experiments with the above medium were performed at different temperatures ranging from 20 'C

to 30 *^C, pH 5.0, aeration rate 0.5-'2.0 wm. Agitation speed was increased gradually fi"om 350 to 1200 rpm to maintain the dissolved oxygen above 30%. Biomass was built up to 300 g/L by 50% glycerol feeding. Aseptically IL broth was harvested and 50 ml of broth was dispensed in each of twelve 250 ml flasks. All flasks were incubated at different temperatures ranging from 20 X to 30 °C at 220 to 260 rpm. 100 jiL of methanol was added every day, and after five days analysis for exendin-4 expression was performed.
At 20 "^C, assay for exendin-4 after five days showed 0.0443 g/L. At 22 °C, assay after five days showed 0.0467 g/L. At 24 °C, assay after five days showed 0.0567 g/L. At 26 'C, assay after five days showed 0.0867 g/L. At 28 °C, assay after five days showed 0.0905 g/L. At 30 ^C, assay after five days showed 0.0685 g/L
Example 8-Fermentation of recombinant Pichia for production of exendin-4
The frozen (-70 ^C) cells oiPichia sp are cultivated in a 250 ml flask containing 50 ml growth medium (1% yeast extract, 2% peptone, 10% IM phosphate buffer (pH 6.0), 0.67% yeast nitrogen base, and 0.1% glycerol) at 28-^32 °C and 220^-260 rpm. The seed cells are cultivated in a 2 L fermenter containing one liter of fermentation medium (3.5% glycerol, 0.01% calcium sulfate, 1% potassium sulfate, 1% magnesium sulfate, 0.25% potassium hydroxide). Fermenter experiments with the above medium were performed at different temperatures ranging from 20 **€ to 30 **€, pH 5.0, aeration rate 0.5'-2.0 wm. Agitation speed was increased gradually from 350 to 1200 rpm to maintain the dissolved oxygen above 30%. Biomass was built up to 285 g/L by 50% glycerol feeding, and then methanol feeding was carried out.
Samples were taken every 24 hour after methanol feeding, and exendin-4 levels were determined using HPLC. At 20 °C, after five days exendin-4 assay showed 0.23 g/L. At 22 °C, after five days assay showed 0.37 g/L. At 24 ^'C, after five days assay showed 0.67 g/L. At 26 °C after five days assay showed 0.72 g/L. At 28 T, after five days assay showed 0.75 g/L. At 30 °C, assay showed 0.67 g/L.
Example 9-Purification of exendin-4

Exendin-4 at a concentration of 300 mg/L was loaded onto a CM-Sepharose cation exchange column at pH 4, and the peptide was eluted at pH 5. The recovery was approximately 65%, and the purity was approximately 80%.
In a subsequent reverse phase (C8) HPLC step, pore size 13 ^, using an acetonitrile-water gradient from 20 to 60%, the exendin-4 peptide was eluted with a purity of 95.5%. The recovery in this step was 20%.
ESI-MS of the purified peptide showed the exact mass of exendin-4 which is calculated to be 4424.66, and N-terminal sequencing indicated the sequence to be HGEGTFTSDL (SEQ ID NO: 8), which is the known sequence of exendin-4.
Example 10-Altemative purification of exendin-4
Exendin-4 at a concentration of 300 mg/L was loaded onto a SP-Sepharose cation exchange column at pH 3.8, and the peptide was eluted at pH 5.4. The recovery was approximately 61%, and the purity was approximately 80%.
In a subsequent reverse phase (C8) HPLC step, pore size 13 /x, using acetonitrile-water gradient from 30 to 50%, the exendin-4 peptide was eluted with a purity of 97.5%. The recovery in this step was 20%.
ESI-MS showed the exact mass of exendin-4 which is calculated to be 4424.66, and the N-terminal sequencing indicated the sequence to be HGEGTFTSDL (SEQ ID NO: 8), which is the known sequence of exendin-4.
Example 11-Peptide mass fingerprinting using trypsin digestion
After partial digestion using trypsin followed by analysis using ESI-MS, four observed masses as mentioned below correlated well to the sequences shown. After the data acquisition, data was converted to Mascot file and uploaded on MATRIXSCIENCE website for peptide mass finger printing for identification. The complete sequence of the expressed exendin-4 peptide could be unambiguously determined.
Observed Mass Sequence correlated

1278.83 HGEGTFTSDLSK (SEQ ID NO: 9)
991.65 QMEEEAVR (SEQ ID NO: 10)
948.83 LFIEWLK (SEQ ID NO: 11)
1082.63 NGGPSSGAPPPSG (SEQ ID NO: 12)
Example 12-C-termmal alpha amidation of exendin-4
The 40 amino acid peptide was converted to an amidated 39 amino acid exendin-4 with the loss of the C-terminal glycine and amidation of the penultimate serine. This step utilized a C-terminal alpha amidating enzyme.
The optimum pH of the enzymatic reaction is 5.4. The reaction was conducted at a temperature of 35 °C. The components in the reaction mixture were 8 mg/ml of exendin-4, 750 units/ml of the amidating enzyme, 8 |aM/ml of catalase, 25 |iM of copper sulfate, and 2 g/L of L-ascorbic acid. The reaction was completed in four hours with an overall yield of 60% of the amidated exendin-4.
Example 13-Alternative C-terminal alpha amidation of exendin-4
The 40 amino acid peptide was converted to an amidated 39 amino acid exendin-4 with the loss of the C-terminal glycine and amidation of the penultimate serine. This required the use of a C-terminal alpha amidating enzyme.
The optimum pH of the enzymatic reaction is 6. The reaction was conducted at a temperature of 37 °C. The components in the reaction mixture included 10 mg/ml of exendin-4,1500 units/ml of the amidating enzyme, 10 |iM/ml of catalase, 25 |aM of copper sulfate, and 1.2 g/L of L-ascorbic acid. The reaction was completed in four hours with an overall yield of 63% of amidated exendin-4.
Example 13-Biological assay of exendin-4
The rat insulinoma cell line Rinm5F was used for these studies. This cell line expresses the GLP-1 receptor to which exendin-4 binds and induces insulin secretion. The secreted insulin was measured by ELISA. The basal insulin secretion was around 3 to 5 pmol/mg whole cell protein. After treatment with exendin-4 for 1 hour, the

insulin secretion by these cells increased from 18 to 28 pmol/mg whole cell protein in three independent experiments.
Other Embodiments
The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims
What is claimed is:
1. A method of producing insulinotropic peptide having biological activity, the
method comprising steps of:
providing a host cell transformed with a polynucleotide vector encoding the polypeptide with insulinotropic activity; and
growing the host cell under suitable conditions to produce the polypeptide.
2. The method of claim 1, wherein the step of providing comprises transforming a host cell with a vector comprising a polynucleotide encoding the polypeptide with insulinotropic activity.
3. The method of claim 1 further comprising the step of isolating the polypeptide.
4. The method of claim 3, wherein the step of isolating the polypeptide comprises isolating the polypeptide from the medium the host cells are growing in,

6. The method of claim 3 further comprising alpha-amidating the polypeptide.
7. The method of claim 1, wherein the cells are yeast cells.
8. The method of claim 1, wherein the cells are Saccharomyces cerevisiae.
9. The method of claim 1, wherein the cells are Pichiapastoris.
10. The method of claim 1, wherein the polypeptide is selected from the group consisting of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and an exendin peptide.

11. The method of claim 1, wherein the polypeptide is exendin-4.
12. The method of claim 1, wherein the host cell is Pichiapastoris, and the polypeptide is exendin-4.
13. The method of claim 11 or 12, wherein the exendin-4 polypeptide is not glycosylated by the host cell.
14. The method of claim 11 or 12, wherein the exendin-4 polypeptide has the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2).
15. The method of claim 1, wherein the polynucleotide vector is a plasmid vector.
16. The method of claim 12, wherein the polynucleotide vector comprises a promoter selected from the group consisting of AOXl and A0X2.
17. The method of claim 1, wherein the step of growing the cells comprises growing the cells under conditions which select for the presence of the polynucleotide vector.
18. The method of claim 1, wherein the step of growing the host cells comprises growing the cells under conditions which induce the expression of the polypeptide.
19. The method of claim 18, wherein the step of growing the host cells comprises adding methanol to the host cells.
20. A polynucleotide encoding exendin-4 optimized for expression of exendin-4 in Pichiapastoris.

Zl. ine polynucleotide of claim 20, wherein the amino acid sequence of exendin-4 comprises HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2).
22. The polynucleotide of claim 20, wherein the amino acid sequence of exendin-4 comprises HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 3).
23. The polynucleotide of claim 20, wherein the sequence comprises
CTC GAG AAA AGA CAT GGA GAA GGA ACA TTT ACA TCT GAT TTG TCT AAA GAA ATG GAA GAA GAA GOT GTT AGA TTG TTT ATT GAA TGG TTG AAA AAC GGA GGA CCA TCT TCT GGA GCT CCA CCA CCA TCT GGA TAA GAA TTC (SEQ ID NO: 5).
24. The polynucleotide of claim 20 further comprising a sequence encoding a signal peptide.
25. The polynucleotide of claim 24, wherein the signal peptide is Mat alpha signal peptide or exendin-4 signal peptide.
26. The polynucleotide of claim 20 further comprising a sequence encoding a propeptide.
27. The polynucleotide of claim 26, wherein the propeptide is Mat alpha propeptide or exendin-4 propeptide.
28. The polynucleotide of claim 20 further comprising a sequence encoding a signal peptide and a propeptide.

^ 29. The polynucleotide of claim 20 further comprising a sequence encoding a purification tag.
30. A vector comprising a sequence encoding exendin-4 operable linked to a promoter.
31. The vector of claim 30, wherein the vector is a plasmid.
32. The vector of claim 30, wherein the vector is replicated in Pichia pastoris.
33. The vector of claim 30, wherein the vector provides for expression of the encoded exendin-4 peptide in Pichia pastoris.
34. The vector of claim 30, wherein the promoter is the AOX promoter.
35. The vector of claim 30, wherein the vector is derived from a vector selected from the group consisting of pPIC, pPIC3K, pPIC9K, and pHIL-D.
36. The vector of claim 30, further comprising a polynucleotide encoding a signal peptide.
37. A Pichia pastoris cell comprising a vector encoding exendin-4.
38. The cell of claim 40, wherein the vector is a vector of one of claim 30-37.
39. An exendin-4 polypeptide of the sequence:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 3),
wherein the C-terminal serine residue is amidated.
40. The exendin-4 polypeptide of claim 39, wherein the polypeptide is not
glycosylated.

41. A pharmaceutical composition comprising exendin-4 and a pharmaceutically
acceptable excipient.
42. A pharmaceutical composition comprising exendin-4 prepared by the method
of claim 1 and a pharmaceutically acceptable excipient.
43. A method of treating a patient with diabetes or preventing hyperglycemia, the
method comprising steps of:
administering to a patient a pharmaceutically acceptable amount of exendin-4.
44. A method of reducing food intake in a subject, the method comprising steps of:
administering to a subject a pharmaceutically acceptable amount of exendin-4.
45. A method of alpha-amidating an exendin-4 polypeptide, the method comprising
steps of:
providing an exendin-4 polypeptide; and
contacting the polypeptide with an C-terminal alpha-amidating enzyme under suitable conditions to yield an alpha-amidated exendin-4 polypeptide.
46. The method of claim 45, wherein the exendin-4 polypeptide has the sequence:
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSG (SEQ ID NO: 2);
and wherein the alpha-amidated exendin-4 polypeptide has the sequence: HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 3), wherein the C-terminal serine is amidated.

Documents:

352-CHE-2004 AMANDED PAGE OF SPECIFICATION 13-11-2009.pdf

352-CHE-2004 EXAMINATION REPORT REPLY RECIEVED 13-11-2009.pdf

352-CHE-2004 OTHER DOCUMENT 13-11-2009.pdf

352-che-2004-abstract.pdf

352-che-2004-claims.pdf

352-che-2004-correspondnece-others.pdf

352-che-2004-description(complete).pdf

352-che-2004-description(provisional).pdf

352-che-2004-form 1.pdf

352-che-2004-form 26.pdf

352-che-2004-form 3.pdf

352-che-2004-form 5.pdf

352-che-2004-other documents.pdf

« Previous Patent

Next Patent »

Patent Number

239944

Indian Patent Application Number

352/CHE/2004

PG Journal Number

17/2010

Publication Date

23-Apr-2010

Grant Date

13-Apr-2010

Date of Filing

19-Apr-2004

Name of Patentee

BIOCON LIMITED.

Applicant Address

20 TH KM HOSUR ROAD, ELECTRONICS CITY P.O. BANGALORE-560100

Inventors:

#	Inventor's Name	Inventor's Address
1	SRIRAM AKUNDI VENKATA	20 TH KM HOSUR ROAD, ELECTRONICS CITY P.O. BANGALORE-560100
2	SURYANARAYANAN SHRIKUMAR	20 TH KM HOSUR ROAD, ELECTRONICS CITY P.O. BANGALORE-560100
3	MELARKODE RAMAKRISHNAN	BIOCON LIMITED,OF 20 TH KM HOSUR ROAD, ELECTRONICS CITY P.O. BANGALORE-560100
4	SASTRY KEDARNATH NANJUND	20 TH KM HOSUR ROAD, ELECTRONICS CITY P.O. BANGALORE-560100

PCT International Classification Number

A61K 38/17

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA