WO2018178796A1 - Method for making kex1 protease sensitive polypeptides using yeast strain - Google Patents

Abstract

Disclosed are methods and compositions for making KEX1 protease-sensitive heterologous polypeptides in yeast, which utilize nucleic acids that encode precursor forms of the polypeptides that contain, at their C-termini, at least one additional amino acid each of which is susceptible to degradation by carboxypeptidase B, and wherein the C-terminal amino acid of the precursor is resistant to degradation by KEX1. Precursor polypeptides that are biologically inactive may then be converted to the mature, biologically active polypeptides via cleavage of the at least one C-terminal amino acid using carboxypeptidase B. Precursors of the polypeptide that are biologically active may be formulated and used to treat diseases such as metabolic disorders. Also disclosed are the precursor forms of the heterologous polypeptides, and nucleic acids encoding them, and genetic constructs containing same.

Description

METHOD FOR MAKING KEX1 PROTEASE SENSITIVE

POLYPEPTIDES USING YEAST STRAIN

Related Application

This application claims the benefit of the Indian Provisional application number 201721010840 filed on March 27, 2017; each of which is hereby incorporated by reference in its entirety.

Field of the Invention

The present invention relates to novel methods and compositions for producing KEX1 protease-sensitive heterologous polypeptides in yeast.

Background of the Invention

Yeast have been the focus of genetic engineers as hosts for production of heterologous proteins. As hosts, they offer several advantages such as rapid growth, genetic background, established fermentation technology and simple media components, a post- translational modification pattern as in eucaryotes, and the ability to secrete the heterologous polypeptides. Thus, yeast may be regarded as an ideal host for producing large quantities of recombinant biopharmaceuticals. Several yeast expression systems are available like Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia, etc. Saccharomyces cerevisiae, a particularly well characterized eucaryotic model organism, has become an attractive host cell for producing recombinant heterologous polypeptides.

On the other hand, yeast produces several proteases which lead to proteolytic degradation of the expression product. A number of endogenous yeast proteases degrade yeast heterologous protein degradation including KEX1, PEP4 and YPS3.

Killer expression-defective protein 1 (KEXl), also called pheromone-processing carboxypeptidase KEXl, is a yeast protease involved in the processing of C-terminal lysine and arginine residues from the endogenous precursors of Kl, K2 and K28 killer toxins and alpha-factor (mating pheromone) (Cooper et ah, 1989). Thus, expression of a heterologous polypeptide in yeast with a C-terminal lysine, arginine or glycine residue also results in C- terminal cleavage of the lysine, arginine or glycine amino acid. This undesired proteolysis of heterologous polypeptides expressed in yeast may lower the desired product yield. It also complicates the downstream processing of the intact product because the degradation products have similar physicochemical and affinity properties. This shortcoming is especially problematic in the case of various biologically active polypeptides such as glue agon-like-pep tide- 1 (GLP-1) and derivatives and analogues thereof (e.g., GLP-1 agonists/incretin mimetics such as liraglutide).

To circumvent the problem of proteolysis by KEX-1 and other endogenous yeast proteins, several approaches have been taken by researchers including engineering of fermentation process, vector systems and host strains (Jones, Methods Enzymol. 94:428-53 (1991); Idris, et al, Appl. Microbiol. Biotechnol. 86(2):403-Π (2010)). Protease-deficient strains are also being used (Gleeson, et al, Methods Mol. Biol. 103:81-94 (1998); Ni, et al, Yeast. 25: 1-8(2008)). For example, Wu, et al, J. Ind. Microbiol. Biotechnol. 40(6) 5%9-99 (2013) showed that disruption of the YPS 1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in yeast. Previously, Sreenivas, et al, Protein Expr. Purif. 118: 1-9 (2016) have shown that disruption of KEXl gene reduces the proteolytic degradation of secreted two-chain insulin glargine in yeast.

The use of non-functional KEXl gene for the expression of heterologous polypeptides has been disclosed (EP0341215; U.S. Patent Nos. 6,103,515 and 8,815,541). However there are several proteases involved in yeast secretion pathway, which present further complications due to the involvement of many cross -reacting factors. Thus, optimizing heterologous polypeptide expression cannot be achieved simply by knocking out just a single protease. Besides, it has been reported that protease deficient strains show typically slower growth rates, lower transformation efficiencies and reduced viability (Lin-Cereghino, et al, Methods Mol. Biol. 389: 11-26 (2007)).

Summary of the Invention

The present invention pertains to methods and compositions of matter for making KEXl protease- sensitive heterologous polypeptides in yeast strains.

The present invention offers a solution to the problem faced by the art without having to knock out or even disable or down-regulate endogenous yeast protease genes. Rather, it entails transformation of yeast with heterologous nucleic acid that encodes a precursor of a heterologous, biologically active polypeptide that is not susceptible to cleavage of C-terminal amino acid residues by KEXl. The precursor heterologous polypeptide includes at least one additional C-terminal amino acid which is not sensitive to cleavage by endogenous yeast proteases, particularly KEXl. The precursor heterologous polypeptide is also designed to facilitate efficient post-translational cleavage of the at least one additional C-terminal amino acid residue by carboxypeptidase B, which results in formation of the mature, heterologous polypeptide.

Accordingly, a first aspect of the present invention is directed to a method of producing a heterologous polypeptide in yeast, wherein the polypeptide is sensitive to KEX1 degradation. The method comprises transforming a KEX1 -functional yeast cell with a heterologous polynucleotide that comprises a nucleic acid having a sequence that encodes a precursor of the heterologous polypeptide which in its mature form has a C-terminal amino acid that is susceptible to degradation by KEX1 (e.g., glycine, arginine and lysine). The nucleic acid comprises (or consists of) at its 3 ' end, an oligonucleotide (also referred to herein as a fragment or tail) containing (or consisting of) one or more codons wherein the 3 ' codon in the oligonucleotide encodes an amino acid that is resistant to degradation by KEX1 but which is susceptible to degradation by carboxypeptidase B (which in some embodiments is a tyrosine (TYR) residue), and wherein each amino acid encoded by the 3 ' oligonucleotide fragment is susceptible to cleavage by carboxypeptidase B. The yeast cell is cultured under conditions suitable for expression of the nucleic acid, thus producing the precursor polypeptide. In embodiments where the precursor polypeptide expression product is biologically inactive, it is contacted with carboxypeptidase B whereby the at least one C- terminal amino acid residue is cleaved, resulting in the mature form of the heterologous polypeptide.

A second aspect of the present invention is directed to a culture of yeast cells in a suitable medium, wherein the yeast cells are KEX1 -functional yeast cells and comprise the heterologous polynucleotide. In some embodiments, the yeast cells further comprise a heterologous polynucleotide comprising a nucleic acid having a sequence that encodes a carboxypeptidase B. In other embodiments, the medium comprises the carboxypeptidase B.

A third aspect of the present invention is directed to a composition comprising the precursor heterologous polypeptide and a carboxypeptidase B. In some embodiments, the composition further includes the fermentation broth or medium used for cultivation of the yeast. In other embodiments, the composition further includes (new) medium. In some embodiments, the composition is substantially free of yeast cells.

Further aspects of the present invention are directed to the precursors of the heterologous polypeptides, per se. In some embodiments, the precursor polypeptides have been surprisingly and unexpectedly found to be biologically active. One particular embodiment is GLP-1 (7-37)-TYR or GLP-1 analogue (2357/05). Accordingly, related aspects of the invention pertain to methods of making GLP-1 (7-37)-TYR or GLP-1 analogue (2357/05), which do not require the post-translational step of cleaving the C-terminal TYR residue, and methods of using this polypeptide to treat Parkinson's disease or metabolic disorders such as type II diabetes and obesity.

Even further aspects of the present invention are directed to genetic constructs, including the heterologous polynucleotides, per se, expression cassettes containing the polynucleotides, vectors containing the expression cassettes, and yeast cells transformed with the vectors.

Brief Description of the Drawings

Figure 1 is a schematic diagram of a plasmid vector useful is the present invention wherein P_Gall is galactose inducible promoter; GOI is the gene of interest; M_URA3 is the URA3 gene encodes orotidine-5' phosphate decarboxylase, an enzyme that is required for the biosynthesis of uracil; Ori_lum is an origin of replication which allow Saccharomyces cerevisiae cells to maintain 20-50 copies of the vector; M_Ampicillin-r is selection marker for E. coli; Ori_pUC is the origin of replication for E. coli.

Figure 2 is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 5P by carboxypeptidase B over the course of a 16-hour incubation.

Figure 3 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PM by carboxypeptidase B over the course of a 16-hour incubation.

Figure 4 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PY by carboxypeptidase B over the course of a 16 -hour incubation.

Figure 5 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PR by carboxypeptidase B over the course of a 16 -hour incubation.

Figure 6 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PL by carboxypeptidase B over the course of a 16-hour incubation. Figure 7 is a graph that shows is a graph that shows percent cleavage of C-terminal amino acid residue of pentapeptide 6PK by carboxypeptidase B over the course of a 16 -hour incubation.

Figure 8 is a graph that shows percent cleavage of C-terminal amino acid residue of various polypeptides produced in yeast, by carboxypeptidase B over the course of a 6-7-hour incubation.

Figure 9 is a graph that shows percent conversion of GLP-1 analogues (sequence id 1), 1532/2357/03 (sequence id 2) and 1532/2357/04 (sequence id 3) by carboxypeptidase B over the course of a 6-hour incubation.

Figure 10 is a graph that shows percent conversion of GLP-1 analogue and a precursor thereof, GLP-1 analogue-TYR, by carboxypeptidase B over the course of a 6-hour incubation.

Figure 11 is a graph that shows change in blood glucose in blood collected from rats over time at various doses of liraglutide, GLP-1 analogue (2357/05) and Victoza®.

Figures 12A and B are graphs showing concentration of GLP-1 analogue (2357/05) and Victoza®, respectively, in blood collected from rats injected with 0.5 mg/kg, as a function of time.

Figures 13A and B are graphs showing concentration of GLP-1 analogue (2357/05) and Victoza®, respectively, in blood collected from rats injected with 1.0 mg/kg, as a function of time.

Detailed Description of the Invention

As used herein, "KEX1" or KEXlp refers to a serine carboxypeptidase, which is endogenous to yeast, that catalyzes the C-terminal cleavage of lysine, arginine and glycine residues. By "functional" KEX1 gene, it is meant the endogenous, unmodified KEX1 gene or a disabled or down-regulated form thereof that still possesses non-negligible enzymatic activity (as compared to a KEX1 "knock out").

As used herein, a precursor polypeptide is a polypeptide that includes at least one additional amino acid at its C-terminus of a mature, biologically active polypeptide, wherein the C-terminal amino acid of the precursor is not sensitive to KEX1 protease but wherein the at least one amino acid is sensitive to cleavage by carboxypeptidase B. As used herein, enzymatic cleavage refers to the hydrolysis or breakage of the polypeptide chains at specific sites within the polypeptide or at the terminal thereof into smaller peptides or amino acids, which are catalysed by enzymes called proteases. One such protease is Carboxypeptidase B which catalyzes the hydrolysis of C-terminal tyrosine, lysine, valine, leucine, isoleucine, asparagine, glutamine and arginine residues from the polypeptides.

As used herein, "gene disruption" or "gene knock-out" refers to the replacement of the functional gene by an inactive gene or deletion of the functional gene. This can be achieved by homologous recombination and/or antisense technology and/or lambda red recombinase and/or CRISPRE and/or zinc finger nuclease technology, and/or regulation of the gene using antisense technology.

As used herein, "heterologous expression" refers to the expression of a nucleic acid in a yeast host cell which naturally does not have the nucleic acid, i.e., the nucleic acid is non- native to the yeast.

As used herein, transformation is a process by which exogenous DNA or nucleic acid is introduced into cells. Such methods are described, for example, in U.S. Patent Nos. 4,845,075 and 4,599,311.

Generally, the heterologous polynucleotide containing the nucleic acid encoding the precursor polypeptide is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression of the nucleic acid. The vector is then introduced into the yeast host through standard techniques. Generally, it will be necessary to select for transformed host cells.

The nucleic acid having the sequence encoding the precursor polypeptide may be operably linked to a DNA sequence encoding a promoter functional in the yeast, a leader sequence and/or other DNA sequences that are necessary for the precursor heterologous polypeptide to be expressed in and secreted from the yeast.

Suitable promoters for S. cerevisiae include the MFal promoter, galactose inducible promoters such as GAL1, GAL7 and GAL 10 promoters, glycolytic enzyme promoters including TPI and PGK promoters, TRP1 promoter, CYCI promoter, CUP1 promoter, PH05 promoter, ADH1 promoter, and HSP promoter. A suitable promoter in the genus Pichia is the AOX1 (methanol utilization) promoter. The DNA constructs that are used for providing secretory expression of the desired polypeptide comprise a DNA sequence that includes a leader sequence linked to the polypeptide by a yeast processing signal. The leader sequence contains a signal peptide ("pre- sequence") for protein translocation across the endoplasmic reticulum and optionally contains an additional sequence ("pro-sequence"), which may or may not be cleaved within yeast cells before the polypeptide is released into the surrounding medium. Useful leaders are the signal peptide of mouse a-amylase, S. cerevisiae MF1, YAP3, BAR1, HSP150 and S. kluyveri MFa signal peptides and prepro-sequences of S. cerevisiae MFal, YAP3, PRC, HSP150, and S. kluyveri MFa and synthetic leader sequences described in WO 92/11378, WO 90/10075 and WO 95/34666.

The transcription terminal signal may include the 3' flanking sequence of a eukaryotic gene which contains proper signal for transcription termination and polyadenylation. Suitable 3' flanking sequences may, e.g., be those of the gene naturally linked to the expression control sequence used, i.e., corresponding to the promoter.

Yeast plasmid vectors that may be useful in the practice of the present invention include the POT (Kjeldsen et al, Gene. 7010-112 (1996)) and Yepl3, Yep24 (Rose et al, Methods Enzymol. 85:234-279 (1990)), and pG plasmids (Schena et al, Methods Enzymol. 94:289-398 (1991)). If integration is desired, the exogenous nucleic acid is inserted into an yeast integration plasmid vector, such as pJJ215, pJJ250, pJJ236, pJJ248, pJJ242 (Jones & Prakash, Yeast 6: 363 (1990)) or pDP6 (Fleig et al, Gene 46:231 (1986)), in proper orientation and correct reading frame for expression, which is flanked with homologous sequences of any non-essential yeast genes, transposon sequence or ribosomal genes. Flanking sequences may include genes used as a selective marker. The DNA is then integrated into host chromosome(s) by homologous recombination occurred in the flanking sequences, by using standard techniques show in Rothstein (Method in Enzymol. 794:281- 301 (1991)) and Cregg et al., (Bio/Technol. 77:905-910 (1993)).

The yeast host cell into which the nucleic acid vector is introduced may be any yeast cell which is capable of expressing the polypeptide. Representative examples include Saccharomyces spp. Or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Further examples of suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansemula, e.g., H. polymorpha, or Pichia, e.g., P. pastoris (cf. Gleeson, et al, J. Gen. Microbiol. 732:3459-3465 (1986); U.S. Patent 4,882,279). The KEX1 gene may be unmodified or down-regulated. In some embodiments, the yeast is modified in that one of more of the endogenous protease genes e.g., YAP3, STE13, PRBl, PEP4, YPS3, YPSl, MKC7, YPS5, YPS6, and YPS7, are down-regulated or knocked out.

Methods for transforming yeast cells with heterologous nucleic acid (DNA) and producing heterologous polypeptides therefrom are well known in the art, as described, e.g., in U.S. Patent Nos. 4,599,311, 4,931,373, 4,870,008, 5,037,743 and 4,845,075. Methods for the transformation of S. cerevisiae include the spheroplast transformation, lithium acetate transformation, and electroporation, cf. Methods in Enzymol. 194 (1991). Transformed yeast cells may be selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g., leucine. Host cells that have been transformed by the exogenous DNA invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression and optimally secretion of the precursor heterologous polypeptides.

The nucleic acid (DNA) sequence encoding the desired mature heterologous peptide may be of genomic or cDNA origin, for instance be obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Patent 4,683,202 or Saiki et al., Science 239:487-491 (1988). The nucleic acid encoding the precursor heterologous polypeptide can then be prepared by joining, e.g., ligating, the oligonucleotide tail to the 3' end of the nucleic acid prepared above. The DNA sequence encoding the heterologous precursor polypeptide may also be prepared synthetically by established standard methods, e.g., the phosphoamidite method described by Beaucage et al., Tetrahedron Letters 22:1859-1869 (1981), or the method described by Matthes et al, EMBO Journal 3:801-805 (1984).

Heterologous polypeptides of interest, especially biologically active polypeptides having C-terminal lysine, arginine and glycine residues (and their corresponding DNA sequences), which can be advantageously produced in accordance with the present methods, are known in the art. Representative polypeptides include pancreatic polypeptide and its analogs, amylin and amylin analogs, PP and analogs, PYY and analogs, oxytocin, vasopressin, incretins such as GLP- 1 and its derivatives and analogues (e.g. , GLP- 1 agonists/incretin mimetics such as liraglutide (marketed by Novo Nordisk under the tradename Victoza®), Lixisenatixe (C-terminal Lys), Albiglutide (C-terminal Lys), Dulaglutide (C-terminal Gly) and Semaglutide (C-terminal Gly)), secretin, calcitonin, gastrin, NPY, FMRF amide, GRHR, CRF, neurokinin A, gastrin releasing peptide, insulin and analogs, and alpha-MSH.

The nucleic acids encoding these polypeptides may be modified by adding to the mature coding sequence a 3 ' oligonucleotide that encodes at least one amino acid that is not susceptible to degradation by KEX1 but which is susceptible to carboxypeptidase B. The 3 ' tail may encode more than one amino acid provided that that C-terminal amino acid of the precursor polypeptide is an amino acid residue that is not susceptible to degradation by KEX1 but is susceptible to degradation by carboxypeptidase B. In preferred embodiments, the 3 ' codon encodes tyrosine. The present inventors have surprisingly and unexpectedly discovered that the carboxypeptidase B cleaves the C-terminal tyrosine residues of polypeptides.

Broadly, the 3 ' oligonucleotide fragment may encode a C-terminal oligopeptide having, for example, 1- 10 amino acid residues and in some embodiments, 1-5 amino acid residues and in some other embodiments, 1-3 amino acid residues and in some other embodiments, 1 amino acid residue. The 3 ' oligonucleotide may thus include codons for amino acids valine, leucine, isoleucine, asparagine, glutamine, lysine, arginine and tyrosine, arranged in appropriate order such that it is not susceptible to degradation by KEX1 (i.e., the C-terminal amino acid is not cleavable by KEX1), and that upon contact with carboxypeptidase B, the mature, biologically active heterologous polypeptide is produced (i.e., each of the at least one amino acids is cleavable by carboxypeptidase B). Representative additional embodiments of the at least one C-terminal amino acid may have the following amino acid sequences:

Leu-Val-Arg-Gly-Arg-Gly-Tyr;

Val-Arg-Gly-Arg-Gly-Tyr;

Lys-Arg-Tyr;

Arg-Lys-Tyr;

Arg-Tyr; and

Lys-Tyr. Thus, a representative example of a polynucleotide encoding a precursor polypeptide of the present invention contains a 3 ' trinucleotide tail encoding tyrosine, is as follows:

GLP1 analog (amino acid seq id 1) + Y

CATGCTGAAGGTACTTTTACTTCTGATGTCTCCTCATACTTGGAAGGTCAAGCTG CTAAAGAATTTATTGCTTGGTTGGTTAGAGGTAGGGGT + TAC/TAT

Carboxypeptidase B is a pancreatic, zinc-containing exopeptidase (EC 3.4.17.2) which as of the time the present invention was made, was known to cleave (via hydrolysis) peptide linkages at basic amino acids, such as lysine, arginine and ornithine, at the C-termini of the polypeptides. Applicant' s discovery thus makes possible the novel methods and compositions which utilize C-terminal tyrosine residue(s) as the substrate for carboxypeptidase B. The carboxypeptidase may be any suitable natural or recombinant carboxypeptidase such as carboxypeptidase B or a mutated variant thereof. In some embodiments, the yeast cell may also be genetically modified (e.g. , transformed with) with nucleic acid encoding the carboxypeptidase gene (sequences of which are known in the art e.g. , U.S. Patent Application Pubs 2008/0311619 Al and 2005/0142633 Al). The DNA encoding carboxypeptidase B may be associated with a leader sequence to allow for secretion of the enzyme from the yeast cell into the culture medium/milieu (or fermentation broth). In other, preferred embodiments, the precursor polypeptide is secreted from or otherwise isolated from the yeast cell (e.g. , via centrifugation) and then added to a suitable medium along with the carboxypeptidase B.

The working examples below provide examples of suitable operating conditions and parameters for enzymatic action of the carboxypeptidase B on the precursor polypeptides. However, the contacting (e.g. , incubation) may be conducted for periods of time ranging from 5 or more minutes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 hours. In general, the amount of enzyme added to a composition containing the precursor polypeptide produced by the yeast cell ranges from about 0.1 units to about 100 units per mg of the precursor polypeptide. As reflected by the sequences above, the at least one additional C-terminal amino acids may include one or more amino acids that is susceptible to cleavage by KEX1, as well as being cleavable by carboxypeptidase B. In addition, for embodiments wherein the C-terminal amino acid of the mature heterologous polypeptide is susceptible to cleavage by carboxypeptidase B, e.g., an arginine or lysine residue, persons skilled in the art will appreciate that the incubation time will have to be selected as necessary so as to terminate the incubation without having this amino acid cleaved via carboxypeptidase B.

Following cleavage of the at least one C-terminal amino acid residue by carboxypeptidase degradation, the mature heterologous polypeptide may be isolated from the composition and purified in accordance with standard techniques.

In other embodiments, cleavage with carboxypeptidase may take place after purification of the precursor polypeptide.

Pharmaceutical compositions containing GLP-1 (7-37)-TYR and Methods of Use

The present invention also related to pharmaceutical compositions comprising GLP-1 (7-37)-TYR or analogues of the present invention and a pharmaceutically acceptable vehicle or carrier.

By GLP-1 (7-37)-TYR, it is meant: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser- Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg/Lys-Gly-Arg- Gly-Tyr.

By GLP-1 analogue (2286/41), it is meant: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-Tyr and at position 26 (Lys) a C16 acyl chain via a glutamyl spacer.

By GLP-1 analogue (2357/05), it is meant: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp- Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-Tyr and at position 26 (Lys) a C16 acyl chain via a glutamyl spacer.

Native GLP-1 (7-37) has a Lys at position 34. Liraglutide differs from native GLP-1 (7-37) in that it has an Arg as position 34, and at position 26 (Lys) a C16 acyl chain via a glutamyl spacer.

GLP-1 (7-37)-TYR may be present in a concentration that is effective to treat the metabolic disease, which generally ranges from about 0.1 mg/ml to about 100 mg/ml, and in some embodiments in a concentration from about 0.1 mg/ml to about 50 mg/ml, and in yet other embodiments in a concentration of from about 0.1 mg/ml to about 10 mg/ml.

GLP-1 analogue (2357/05) may be present in a concentration that is effective to treat the metabolic disease, which generally ranges from about 0.1 mg/ml to about 100 mg/ml, and in some embodiments in a concentration from about 0.1 mg/ml to about 50 mg/ml, and in yet other embodiments in a concentration of from about 0.1 mg/ml to about 10 mg/ml. The pharmaceutical compositions may include an isotonic agent and/or a buffer. Examples of isotonic agents include sodium chloride, mannitol and glycerol and propylene glycol. The propylene glycol may be present in a concentration that generally ranges from about 1 to about 50 mg/ml, and in some embodiments in a concentration of from about 5 to about 25 mg/ml, and in yet other embodiments in a concentration of from about 8 to about 16 mg/ml.

Propylene glycol-containing compositions may be advantageously formulated to have a pH in the range from about 7.0 to about 9.5. and in some embodiments from about 7.0 to about 8.0, and in some other embodiments from 7.2 to about 8.0, and in some other embodiments from about 7.0 to about 8.3, and in yet further embodiments from 7.3 to about 8.3.

The composition may further contain a preservative, representative examples of which include phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2- phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, and combinations of two or more thereof. The preservative may be present in a concentration that generally ranges from about 0.1 mg/ml to about 50 mg/ml, and in some embodiments from about 0.1 mg/ml to about 25 mg/ml, and in yet other embodiments from about 0.1 mg/ml to about 10 mg/ml.

Representative examples of buffers include sodium acetate, sodium phosphate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, and tris(hydroxymethyl)- aminomethan, and combinations of two or more thereof.

The pharmaceutical compositions may further contain a surfactant in order to improve the solubility and/or the stability of the GLP- 1 (7-37)-TYR or its analogues. Representative examples of surfactants, such as zwitterionic surfactants, cationic surfactants, non-ionic surfactants and polymeric surfactants as well as detergents, that may be suitable for use in the present invention include ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, poloxamers, such as 188 and 407, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, glycerol, cholic acid or derivatives thereof, lecithins, alcohols and phospholipids, glycerophospholipids (lecithins, kephalins, phosphatidyl serine), glyceroglycolipids (galactopyransoide), sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides, gangliosides), DSS (docusate sodium, CAS registry no [577- 11-7]), docusate calcium, CAS registry no [128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), dipalmitoyl phosphatidic acid, sodium caprylate, bile acids and salts thereof and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N-Hexadecyl-N,N-dimethyl-3-ammonio- l- propanesulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, palmitoyl lysophosphatidyl-L-serine, lysophospholipids (e.g. l-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine), alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether)- derivatives of lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, zwitterionic surfactants (e.g. N-alkyl- N,N-dimethylammonio- 1 -propanesulfonates, 3 -cholamido- 1 -propyldimethylammonio- 1 - propanesulfonate, dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egg lysolecithin), cationic surfactants (quarternary ammonium bases) (e.g. cetyltrimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants, polyethyleneoxide/polypropyleneoxide block copolymers (Pluronics/Tetronics, Triton X- 100, Dodecyl β-D-glucopyranoside) or polymeric surfactants (Tween-40, Tween-80, Brij-35), fusidic acid derivatives-(e.g. sodium tauro-dihydrofusidate etc.), long-chain fatty acids and salts thereof C6-C 12(e.g. oleic acid and caprylic acid), acylcarnitines and derivatives, N^a - acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, N^a -acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N^a -acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, or the surfactant may be selected from the group of imidazoline derivatives.

The pharmaceutical compositions may further contain a chelating agent, representative examples of which include salts of ethlenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and combinations of two or more thereof. The chelating agent may be present in a concentration generally ranging from 0.1 mg/ml to 5 mg/ml, and in some embodiments from 0.1 mg/ml to 2 mg/ml, and in yet other embodiments from 2 mg/ml to 5 mg/ml. The pharmaceutical compositions may further contain a stabilizer, representative examples of which include high molecular weight polymers or low molecular compounds e.g. , polyethylene glycol (e.g. PEG 3350), polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxymethylcellulose, salts (e.g. sodium chloride), L-glycine, L-histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and combinations of two or more thereof.

In some embodiments, the high molecular weight polymer is present in a concentration from 0.1 mg/ml to 50 mg/ml, and in other embodiments from 5 mg/ml to 10 mg/ml, and in yet other embodiments from 0.1 mg/ml to 5 mg/ml. In some embodiments, the low molecular weight polymer is present in a concentration from 0.1 mg/ml to 50 mg/ml, and in other embodiments from 5 mg/ml to 10 mg/ml, and in yet other embodiments from 0.1 mg/ml to 5 mg/ml. The pharmaceutical compositions may also contain zinc.

The pharmaceutical compositions may further contain another active agent, e.g., an antidiabetic (e.g., insulin such as human insulin, and oral hypoglycemic agents) and/or an anti-obesity agent (e.g. , leptin, amphetamine, dexfenfluramine, sibutramine, orlistat).

The pharmaceutical compositions of the present invention may be prepared by conventional techniques, e.g., as described in Remington's Pharmaceutical Sciences, 1985. For example, injectable compositions of the present invention can be prepared using the conventional techniques known in the pharmaceutical industry which involves dissolving and mixing the ingredients as appropriate to give the desired end product. A composition for nasal administration may, for example, be prepared as described in European Patent 272097 (to Novo Nordisk A/S) or in WO 93/18785. In some embodiments of the present invention, GLP-1 (7-37)-TYR is provided in the form of a composition suitable for administration by injection. Such a composition can either be an injectable solution ready for use or it can be an amount of a solid composition, e.g. a lyophilised product, which has to be dissolved in a solvent before it can be injected. The injectable solution may contain not less than about 2 mg/ml, and in some embodiments not less than about 5 mg/ml, and in other embodiments not less than about 10 mg/ml and in yet other embodiments, not more than about lmg/ml of GLP-1 (7-37)-TYR or its analogue.

GLP-1 (7-37)-TYR or its analogue and pharmaceutical compositions containing it may be used to treat metabolic diseases, examples of which include non-insulin dependent diabetes mellitus, insulin dependent diabetes mellitus, obesity (including glycemic control) and insulin resistance. The pharmaceutical compositions of the present invention may in some embodiments, be administered parenterally to patients in need of such a treatment. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. In other embodiments, A further option is to formulate GLP-1 (7-37)-TYR in a composition which may be a powder or a liquid for administration via a nasal or pulmonary spray. In further embodiments, GLP-1 (7-37)-TYR may be formulated for transdermal administration e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally. With respect to glycemic control, starting dosages may be about 0.6 mg/day for about one week (to reduce gastrointestinal symptoms), and then about 1.2 mg day, which may be increased to about 1.8 mg/day in the event the lower dose is ineffective.

Working examples

The present invention will now be described in terms of the following non-limiting examples. General molecular biology, recombinant DNA technology, methods and protein purification used throughout the experimental work are as described in J. Sambrook, et ah, Molecular Cloning: A Laboratory Manual, fourth edition, Cold Spring Harbor Laboratory, New York.

Example 1: Enzymatic Cleavage of C-terminal Amino Acid of Pentapeptides

Six pentapeptides, designated 5P, 6PM, 6PY, 6PR, 6PL and 6PK, having the respective amino acid sequences set forth below, were subjected to treatment with carboxypeptidase B. The amino acid sequences of each of the six pentapeptides are as follows:

5P: VRGRG

6PM: VRGRGM

6PY: VRGRGY

6PR: VRGRGR

6PL: VRGRGL

6PK: VRGRGK The reaction protocol was as follows:

Reaction set up procedure for each pentapeptide

1.0 μg enzyme : 100 μg peptide (in 300 μΐ)

Weigh 1.5 mg peptide & transfer in 10 ml capacity glass tube.

Ψ

Dissolve peptide in 4.35 ml of Tris-Cl + 100 mM Nacl pH 7.65 to get 345μg/ml final cone.

Ψ

Take 2610 μΐ of peptide stock (345μg/ml)

Ψ

Add 9 μg enzyme (90 μΐ of 100 μg/ml stock)

Ψ

Prepare six aliquots, 300 μΐ/tube & label as follows for each pentapeptide: Al, A2, A4, A6,

A12, A16

Note: Set up one peptide reaction control without enzyme.

A16 C (290 μΐ of peptide stock + 10 μΐ water)

Ψ

Incubate at 25° Celsius

Ψ

Stop the reaction with lul of 300 mM EDTA stock as per given schedule.

1) Al after 1 hrs

2) A2 after 2 hrs

3) A4 after 4 hrs

4) A6 after 6 hrs

5) A12 after 12 hrs

6) A16 after 16 hrs

7) A16 C after 16 hrs

Post incubation, store I reaction mix at -20° Celsius

Note: Set up one common enzyme reaction control blank without peptide (290 μΐ Tris buffer + 10 μΐ enzyme)

The results are graphically illustrated in Figs. 2-7. The data show that the peptide 6PY, having the C-terminal tyrosine residue (Y), was the most susceptible to degradation by carboxypeptidase B, wherein 100% of the tyrosine residues were cleaved within about 6 hours. See Fig. 4. The data also show that the C-terminal R and K residues, i.e., arginine and lysine, respectively, were also quite susceptible to carboxypeptidase B degradation, although not as susceptible as tyrosine. See, Figs. 5 and 7. On the other hand, proline (P), methionine (M) and leucine (L), were substantially if not totally resistant to carboxypeptidase B degradation. See, Figs. 2, 3 and 6.

Example 2: Yeast Host Cells

The S. cerevisiae yeast host cells were obtained from American Type Cell Culture collection (ATCC Number 4026510). Overnight cultures of S. cerevisiae grown in nonselective YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 25 °C were inoculated at 1: 100 in YPD medium and grown to an optical density at 600 nm (OS600) of 1.6 for competent cell preparation. The culture was chilled on ice for 15 min and centrifuged at 4000g. The cell pellet was treated with lOOmL of TE buffer containing Lithium Acetate for 45 min at 30°C shaking at about 85 RPM 2.5 mL of 1 M DTT was added and incubated for 15 min at 30°C. Cells were pelleted down and washed with ice cold water followed by 1M sorbitol solution.

Example 3: Constructs and Transformation into Yeast

For the expression of the heterologous polypeptides, a pSCOOl vector was used, operably linked to a galactose inducible promoter (Figure 1). The aliquot of 40 μΐ of electro- competent cells was mixed with 1 μg of expression vector in 0.2 cm cuvettes and electroporated using Gene Plaste Xcell (Make: Biorad). Following electroporation, cells were plated directly onto selective agar plate (SD media without uracil, Cat No. G067 Make: Himedia) and the plate was incubated at 25°C for 4 days. The selected colonies were transferred to a flask containing 100 ml SD media and expression was induced with 2% galactose at an optical density 10. Precursor polypeptides were purified with series of chromatography steps.

Example 4: Carboxypeptidase B treatment of heterologous polypeptides

Six heterologous polypeptides were expressed in the S. cerevisiae and purified using the constructs and methods described in earlier examples 2 and 3. The sequences of the six heterologous polypeptides were as follows:

<5P(A)>

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRG <6P(B)>

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRG Y

<6P(C)>

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRGR

<6P(D)>

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRGM

<6P(E)>

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRGL

<6P(F)>

HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK

The expressed heterologous polypeptides (5P-A, 6P-B, 6P-C, 6P-D, 6P-E, 6P-F) were treated to cleave the C-terminal additional amino acid according to the same protocol described in example 1.

The results set forth in are graphically illustrated in Fig. 8. The results show that the polypeptide 6P(B) containing the C-terminal tyrosine residue was the most susceptible to degradation/cleavage by carboxypeptidase B - substantially 100% of the C-terminal tyrosine residues were cleaved within about 4 hours.

Example 5: Cleavage of C-Terminal Amino Acid from Precursor Liraglutide

We followed the protocol for enzymatic cleavage of peptides given in example 1. Figure 9 shows percent conversion of GLP-1 analogue (sequence id 1), 1532/2357/03 (sequence id 2) and 1532/2357/04 (sequence id 3) by carboxypeptidase B over the course of a 6-hour incubation. Figure 10 shows percent conversion of GLP-1 analogue and a precursor thereof, GLP-1 analogue-TYR, by carboxypeptidase B over the course of a 6-hour incubation. Seq id 1 (GLP-1 analog)

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRG

Seq id 2 (1532/2357/03)

HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK

Seq id 3 (1532/2357/04)

H AEGTFTS D VS S YLEGQ A AKEFIA WLVRGRG Y

Example 6: Oral Glucose Tolerance Test (OGTT) in normal rats

Glucagon like peptide 1 (GLP- 1) is a metabolic hormone that stimulates a decrease in blood glucose level. GLP-1 consists of 30 amino acids and it is derived from tissue-specific post-translational processing of the proglucagon gene located on chromosome 17. GLP- 1 further undergoes post-translational amidation at C-terminus. This amidation and the histidine residue at position 7 are very important for its activity. Amidation has also been shown to prolong the survival of GLP- 1 in the blood stream (Vishal, Indian J. Endocrinol. Metab. 17(3 ):413-21 (2013). This example shows that GLP- 1 analogue (2357/05) has increased plasma half-life compared to liraglutide or GLP- 1 (7-37).

Female Sprague Dawley rats having body weight of 190-210 gm were divided into 5 groups consisting of 3 rats in each group. All the animals were fasted overnight (16 hours). Group 1 : Normal control received Phosphate Buffer pH 8.0

Group 2: Liraglutide (2286/41, 0.5 mg/kg, s.c.)

Group 3: GLP-1 Analogue (2357/05, 0.5 mg/kg, s.c.)

Group 4: Victoza^® (0.2 mg/kg, s.c.)

Group 5: Victoza^® (0.5 mg/kg, s.c.)

20% Glucose solution was prepared. Baseline blood glucose levels were measured with glucose strips using Blood Glucose Meter (One Touch™ Ultra™; LIFESCAN, Johnson & Johnson). After 4 hours of drug administration, all the animals were dosed orally with the 20% glucose solution at a dose of 2 gm/kg. Approximately 5 μΐ_^ of blood was collected and blood glucose levels were measured with glucose strips using Blood Glucose Meter (One Touch™ Ultra™; LIFESCAN, Johnson & Johnson) at 20, 40, 60, 90 and 120 minutes post oral glucose administration.

The inventive embodiment results are illustrated in Fig. 11. They show that they efficacy of GLP- 1 analogue (2357/05) at C_max = 300 mg/ml was equivalent to a C_max = 600 mg/ml of Victoza®, and that this embodiment exhibited a proportionate dose-dependent C_max response.

Example 7: Pharmacokinetic study in normal rats

Female Sprague Dawley rats having body weight of 190-240 gm were divided into 4 groups consisting of 4 rats in each group. All the animals were fasted overnight (16 hours). Group 1 : GLP-1 analogue (2357/05) (0.5 mg/kg, s.c.)

Group 2: GLP-1 Analogue (2357/05) (1 mg/kg, s.c.)

Group 3: Victoza^® (0.5 mg/kg, s.c.)

Group 4: Victoza^® (1 mg/kg, s.c.)

Approximately 400 μL· of blood was collected by retro-orbital plexus puncture using glass capillaries in labelled (label includes Study number, Animal ID, Time point, Group) micro centrifuge tubes containing anticoagulant (15 μΐ_^ of 10% K₂EDTA) at 0, 0.5, 1, 2, 4, 6, 8, 10 and 24 hours post subcutaneous injection. Plasma was separated from the collected blood samples by centrifugation at approximately at 3000 rpm for 10 min at 4°C and was transferred into other labelled (label includes Study number, Animal ID, Time point, Group) micro centrifuge tube. Plasma was stored at about -70°C (+100°C) in deep freezer, until analysis using LC -MS/MS. The results are illustrated in Figs. 12 and 13 and tabulated in Tables I-IV.

Table I (GLP- 1 analogue 2357/05)

Dose Cmax AUC 0-t AUC 0-inf Tmax Half Life (mg/kg) (ng/mL) (hr*ng/mL) (hr*ng/mL) (hr) (hr)

0.5 311.2 4458.9 5161.6 6.5 8.3 Table II (Victoza®)

Figs. 12A and B show that the plasma half-life of GLP-1 analogue (2357/05) is comparatively higher than Victoza® at the 0.5 mg/ml dose. These data are tabulated in Tables I and II, respectively (for the inventive embodiment and Victoza®).

Figs. 13A and B show that GLP-1 analogue (2357/05) exhibited a concentration in plasma of greater than 100 mg/ml at 24 hours, which is higher than the plasma concentration of Victoza® at 24 hours. These data are tabulated in (GLP-1 analogue 2357/05) Tables III and IV.

Table III (GLP-1 analogue 2357/05)

All publications cited in the specification, including patent publications and nonpatent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporate by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

Claims What is claimed is:

1. A method of producing a heterologous polypeptide in yeast, wherein the polypeptide is susceptible to KEX1 degradation, wherein the method comprises transforming a KEX1- functional yeast cell with polynucleotide comprising a nucleic acid having a sequence that encodes a precursor of the heterologous polypeptide wherein the nucleic acid comprises at its 3' end, an oligonucleotide comprising one or more codons wherein the 3' codon encodes an amino acid that is resistant to degradation by KEX1 but which is susceptible to degradation by an enzyme, and wherein each amino acid encoded by the 3' oligonucleotide is susceptible to cleavage by the enzyme; culturing the yeast cell under conditions suitable for expression of the nucleic acid, thus producing the precursor polypeptide; contacting the precursor polypeptide expressed by the nucleic acid with the enzyme whereby the at least one C- terminal amino acid residue is cleaved, resulting in the mature form of the heterologous polypeptide.

2. The method of claim 1, wherein an endogenous yeast gene encoding the KEX1 is down-regulated.

3. The method of claim 1 or claim 2, wherein the yeast cell is Saccharomyces cerevisiae.

4. The method of claim 1 or claim 2, wherein the enzyme is carboxypeptidase B.

5. The method of any of claims 1-4, wherein the 3' oligonucleotide is a trinucleotide encoding tyrosine, such that the precursor polypeptide differs from the mature heterologous polypeptide in that it contains a C-terminal tyrosine residue, and wherein the enzyme is carboxypeptidase B.

6. The method of any of claims 1-4, wherein the 3' oligonucleotide encodes more than 1 amino acid and wherein the C-terminal amino acid residue encoded by the oligonucleotide is a tyrosine residue.

7. The method of claim 6, wherein the 3' oligonucleotide encodes arginine and tyrosine, such that the precursor polypeptide differs from the mature heterologous polypeptide in that it contains C-terminal arginine and tyrosine residues.

8. The method of claim 6, wherein the 3' oligonucleotide encodes lysine and tyrosine, such that the precursor polypeptide differs from the mature heterologous polypeptide in that it contains C-terminal lysine and tyrosine residues.

9. The method of claim 6, wherein the 3' oligonucleotide encodes glycine and tyrosine, such that the precursor polypeptide differs from the mature heterologous polypeptide in that it contains C-terminal glycine and tyrosine residues.

10. The method of claim 6, wherein the 3' oligonucleotide encodes one or more of lysine, valine, leucine, isoleucine, asparagine, glutamine and arginine residues.

11. The method of any of claims 1-10, wherein the mature heterologous polypeptide has C-terminal glycine, arginine or a lysine residue.

12. The method of any of claims 1-11, wherein the contacting is conducted in culture medium used for the cultivating.

13. The method of claim 12, wherein the carboxypeptidase B is added to the culture medium.

14. The method of claim 12, wherein the yeast cells further comprise an exogenous nucleic acid encoding a carboxypeptidase B, and the carboxypeptidase B expressed by the nucleic acid is secreted by the yeast into the culture medium.

15. The method of any of claims 1-14, wherein the contacting is conducted for a period of time ranging from about 5 minutes to about 16 hours.

16. The method of any of claims 1-15, wherein the mature polypeptide is selected from the group consisting of pancreatic polypeptide and its analogs, amylin and amylin analogs, PP and analogs, PYY and analogs, oxytocin, vasopressin, GLP-1 and analogues, secretin, calcitonin, gastrin, NPY, FMRF amide, GRHR, CRF, neurokinin A, gastrin releasing peptide, insulin and analogs, and alpha-MSH.

17. The method of claim 16, wherein the GLP-1 analog is selected from the group consisting of liraglutide, Lixisenatixe, Albiglutide, Dulaglutide and Semaglutide.

18. A composition comprising yeast cells and a culture medium, wherein the yeast cells are KEXl -functional yeast cells and comprise a heterologous polynucleotide comprising a promoter functional in the yeast cells operably linked to a nucleic acid having a sequence that encodes a precursor of a heterologous polypeptide, the mature form of which is susceptible to cleavage by KEXl, wherein the nucleic acid comprises at its 3' end, an oligonucleotide comprising one or more codons wherein the 3' codon encodes an amino acid that is resistant to degradation by KEXl but which is susceptible to degradation by carboxypeptidase B, and wherein each amino acid encoded by the 3' oligonucleotide is susceptible to cleavage by carboxypeptidase B.

19. The composition of claim 18, further comprising the carboxypeptidase B.

20. A precursor of a polypeptide which in its mature form has a C -terminal amino acid that is susceptible to degradation by KEXl, wherein the precursor includes the mature form of the polypeptide and at least one C-terminal amino acid, each of which is susceptible to degradation by carboxypeptidase B, and wherein the C-terminal amino acid of the precursor is resistant to degradation by KEX1.

21. The precursor polypeptide of claim 20, wherein the C-terminal amino acid residue is tyrosine.

22. The precursor polypeptide of claim 20 or claim 21, wherein the mature form of the polypeptide has a C-terminal lysine, arginine or glycine residue.

23. A composition comprising the precursor polypeptide of any of claims 19-22, and a carboxypeptidase B.

24. A polynucleotide comprising a nucleic acid having a sequence that encodes the precursor polypeptide of any of claims 20-22.

25. An expression cassette comprising the polynucleotide of claim 24.

26. A vector comprising the expression cassette of claim 25.

27. The vector of claim 27, which is a plasmid.

28. A yeast cell transformed with the vector of claim 27.

A polypeptide, which is GLP-1 (7-37)-TYR.

30. The polypeptide of claim 29, which is GLP-1 (7-37, Arg 34)-TYR.

31. A polypeptide, which is GLP-1 analogue (2357/05).

32. A pharmaceutical composition, comprising an amount of the polypeptide of claim 29 or claim 31 effective to treat a metabolic disorder, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutically acceptable carrier comprises propylene gylcol.

34. A method of treating a metabolic disorder, comprising administering the composition of claim 32 to a human patient in need thereof.

35. The method of claim 34, wherein the said metabolic disorder is selected from the group consisting of Type 2 diabetes, obesity, elevated blood pressure, elevated fasting plasma glucose.