Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons

Definitions

• the present invention is related to a method of producing acylated proteins or poly- peptides by expressing certain precursors of the desired polypeptide that protects the expressed polypeptide against proteolytic degradation within the host cell.
• the invention is also related to DNA-sequences, vectors and transformed host cells for use in the claimed method.
• the present invention is related to certain precursors of the desired polypeptides and an acylation method for acylation method for acylation of one or more lysine resi- dues in the desired polypeptide.
• Recombinant DNA technology has enabled expression of foreign (heterologous) polypeptides in microbial and other host cells.
• yeast expression of heterologous polypeptides after transformation of yeast cells with suitable expression vectors comprising DNA se- quences coding for said polypeptides has been successful for many species of polypeptides, such as insulin and insulin precursors, glucagon, glucagon like peptides and analogues thereof.
• a common problem with expression of proteins or polypeptides of a limited size in a recombinant host is, however, proteolytic degradation of the expressed product by proteolytic enzymes produced by the host organism.
• the isolated product may be a heterogeneous mixture of species of the desired polypeptide having different amino acid chain lengths.
• Yeast contains a number of proteases used for processing yeast proteins e.g. Kex2p and Ypsl p which cleave at the C-terminal side of a dibasic amino acid sequence, and the carboxypeptidase Kexlp which digests remaining basic amino acids after the endoproteolytic digestion by Kex2p, and Ste13p or Dap2p which cleave at X-Ala or X-Pro.
• proteases used for processing yeast proteins e.g. Kex2p and Ypsl p which cleave at the C-terminal side of a dibasic amino acid sequence
• carboxypeptidase Kexlp which digests remaining basic amino acids after the endoproteolytic digestion by Kex2p
• Ste13p or Dap2p which cleave at X-Ala or X-Pro.
• polypeptides e.g. polypeptides having from about 10 to about 100 amino acids chains and none or a few disulphide bonds and/or are rich in basic amino acids, such as ⁇ -endorphine, glucagon and glucagon like peptides may be especially susceptible to intracellular and extracellular proteolytic degradation when expressed in a transformed host cell due to their short-chain open and non-disulfide stabilized structure resulting in an inhomogeneous product which may be proteolytically degraded in the N- and C-terminal ends as well as endoproteolytically degraded.
• N-terminal cleavage of expressed polypeptides by host cell produced enzymes may cause decreased yield of a desired product with correct N-terminal if the N- terminal of the expressed product constitutes a cleavage site for endogenous enzymes.
• yeast for example the enzyme Ste13p cleaves at X-Ala or X-Pro, where X can be any amino acid residue.
• polypeptides with an Ala or Pro residue as the second residue from the N-terminal end may be cleaved at the N-terminal end and the recovered polypeptide may be a mixture of different degradation products complicating the recovery process and reducing the overall yield.
• small polypeptides with little tertiary structure and low content of ⁇ - helixes may have a higher tendency to form ⁇ -sheets that stack on each other and form fibrils during fermentation and down stream separation and purification steps in large scale production. Formation of fibrils may cause unwanted precipitation with loss of the desired product. Fibrillation may be prevented by treatment at high pH. However, such alkaline treatment is pretty harsh to the product and may cause unwanted formation of D-amino acids residues.
• Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesised in the L-cells in the distal ileum, in the pancreas and in the brain. Processing of preproglucagon to give GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2 occurs mainly in the L- cells. Both GLP-1 and GLP-2 have an Ala as the second amino acid residue from the N- terminal end and are thus prone for N-terminal cleavage when expressed in a host organism such as yeast.
• the present invention is related to a method for making a polypeptide comprising at least one lysine residue being acylated in its ⁇ -amino group, said method comprising the following steps:
• the order of steps (ii) to (iv) may be changed.
• the acylating step (iii) is conducted after the removal step (iv) so that the N-terminal extension is removed before the polypeptide is acylated in the desired position.
• the desired polypeptide may contain more than one lysine residue as a potential target for acylation but will typically only contain one lysine residue. Thus, in one embodiment, the desired polypeptide is monoacylated.
• the N-terminal extension will typically be of up to 15 amino acids in length and may be from 1-15; 2-15; 3-15; 3-12; 3-10; 3-9; 3-8; 3-7; 3-6; or 3-5 amino acids in length.
• the amino acids in the N-terminal extension are selected with a multiple purpose: 1) to protect the expressed precursor molecule against endoproteolytic degradation; 2) to avoid acylation at the N-terminal amino acid residue of the desired polypeptide, i.e. to ensure that acylation takes place preferentially or only at the wanted position in the desired polypeptide; and 3) to prevent precipitation caused by fibrillation during fermentation and down stream processing steps such as separation and purification in large scale production.
• the amino acid residues at both ends of the N-terminal extension should be selected so as to ensure efficient cleavage of the N-terminal extension from the desired polypeptide at the C-terminal end and at the N-terminal end from possible upstream sequences such as pre- or pre-pro peptides which have the purpose of ensuring transport of the expressed precursor molecule out of the host cell and into the culture medium.
• the N-terminal extension may serve as a tag for purification purposes.
• the N-terminal extension is found to be stably attached to the precursor molecule of the invention during fermentation, protecting the N-terminal end of the precursor molecule against the proteolytic enzymes such as Ste13p and/or Dap2p.
• the N-terminal extension is removably attached to the N-terminal end of the desired polypeptide.
• the C-terminal end of the N-terminal extension will constitute a cleavage site or will together with amino acid residues at the N-terminal end of the desired polypeptide constitute a suitable cleavage site.
• This cleavage site is different from lysine to avoid acyla- tion of the precursor molecule at this position.
• Cleavage may be conducted by means of chemicals like cyanogen bromide (E. Gross: Methods in Enzymlogy XI, 1967, 238-255, Editor: CHW Hirs, and JP Whitelegge et al, Protein Science 2000, 9, 1618-1630) or hydroxylamine cleaving at the C-terminal side of Met or Asn.
• Asn cleavage is enhanced by the presence of a Gly N-terminal to the cleavage site (Asn IGly).
• the cleavage can also be effected by specific exoproteases such as a suitable proteolytic enzyme which is specific for the chosen cleavage site, cleaving at an N-terminal pyroglutamic acid or endoproteases like praline endopeptidases (EC 3.4.22.26) cleaving at the C-terminal side of Pro in polypeptides.
• endoproteases as trypsins cleave at the C-terminal side of many single Arg-residues, while other like Factor Xa is more specific and often cleaves after the sequence lle-Glu-Gly-Arg.
• Kex2p or PC1 and similar enzymes cleave at a dibasic cleavage site such as Arg-Arg.
• a mixture of chemical and enzymatic methods can be used if a Cys residue is placed N-terminally to the polypeptide.
• the SH-group can easily be acylated with an amine that is generating a pseudo-lysine amino acid which can then be cleaved by Achromobacter lyticus protease I after acylation of the lysine residue in the desired polypeptide, thereby protecting this site from cleavage.
• the cleavage site is chosen from the group consisting of Met, Asn, Pro, Gin, Cys and Arg-Arg.
• the desired polypeptide will comprise a Pro, Ala or Ser residue as the second amino acid residue from the N-terminal end.
• Such polypeptides will be especially vulnerable to degradation by proteolytic enzymes such as Ste13p.
• the desired polypeptide will have a His-Ala or a His-Pro, His-Ser or a Tyr as the N-terminal sequence.
• the N-terminal extension has the formula wherein X n X-i is a peptide sequence of from 1 - 14 amino acid residues in length and Y is Met, Asn, Pro, Gin, Cys or Arg-Arg, the function of X n Xi - Y being a) to protect the expressed polypeptide from endoproteolytic cleavage, b) to prevent acylation at the N- terminal end of the desired polypeptide and c) to prevent precipitation caused by fibrillation during fermentation and down stream separation and purification steps.
• the amino acid residues in X n X - Y are furthermore selected to obtain optimal in vitro cleavage of the N- terminal extension at its C-terminal end (at Y) and optimal in vivo cleavage at its N-terminal end (at X n ) at a KEX site from upstream signal-leader sequences.
• the amino acid residues in X n Xi may in principle be any amino acid residue except Lys as long as the peptide sequence fulfils at least one of the required purposes.
• number two amino acid residue from the N-terminal end of the extension is preferably not Ala or Pro.
• X n X ⁇ is a peptide sequence of from 1-15; 2-15; 3-15; 3-12;
• N-terminal amino acid residues in X n Xi are preferably chosen from Glu and Asp. Glu and/or Asp positioned at the N-terminal end of the N-terminal extension will also protect the expressed molecule against proteolytic degradation in the yeast cell.
• N-terminal extensions are Glu-Glu-Met; Glu-Glu-Ala-Glu-Met(SEQ ID NO:1); Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2); Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3); Gin; Glu-Pro-Gln(SEQ ID NO:4); Glu-Ala-Gln; Glu-Ala-Glu-Ala-Gln(SEQ ID NO:5); Glu-Ala-Glu- Ala-Glu-Ala-Gln(SEQ ID NO:6); Glu-Glu-Gly-Cys-Thr-Ser-lle-Cys(SEQ ID NO:7); Glu-His- Gly-Cys-Thr-Ser-lle-Cys(SEQ ID NO:8); Glu-Glu-Ala-Arg-Met(SEQ ID NO:9); Glu-Glu-Arg- Asn(SEQ ID NO:10); Glu-
• the present invention is related to a polypeptide precursor for a desired polypeptide said polypeptide precursor having the formula N-terminal extension-Y-i - * polypeptide *
• Y. is Met, Asn, Pro, Gin, Cys or Arg-Arg; the N-terminal extension has 1-14 amino acid residues as described above and * polypeptide * is the remaining part of thede- sired polypeptide.
• the present invention is related to a polypeptide precursor for a desired polypeptide said polypeptide precursor having the formula
• Y ⁇ is Met, Asn, Pro, Gin, Cys or Arg-Arg; Y 2 is His or Tyr, Y 3 is Ala, Ser or Gly, the N-terminal extension has 1-14 amino acid residues as described above and *polypeptide* is the remaining part of desired polypeptide.
• Y 2 is N-terminal amino acid residue in the desired polypeptide and Y 3 is the second amino acid residue from the N-terminal end of the desired polypeptide
• the present invention is related to polynucleotides encoding the claimed polypeptide precursors and vectors and transformed host cells containing such polynucleotides.
• acylated peptides which have a protracted profile relative to the native peptide or unmodified analogues. This phenomenon is disclosed and demonstrated in WO 98/08871 which discloses acylation of GLP-1 and analogues thereof and in WO 98/08872 which discloses acylation of GLP-2 and analogues thereof.
• the lipophilic group may be introduced by means of mono- or dipeptide spacers as disclosed in WO 98/08871.
• the lipophilic group may be introduced by means of ⁇ -amino- ⁇ , ⁇ -dicarboxylic acid groups as disclosed in WO 00/55119.
• the present polypeptide precursor will contain at least one lysine group with a free ⁇ -amino group to be acylated.
• acylating one or more free amino acid groups in a polypeptide acylation of the free amino group in the N-terminal amino acid residue is more or less avoidable.
• Certain methods have been developed to avoid acylation at the N-terminal amino acid residue, vide US patent No. 5,905,140.
• the present invention offers an alternative solution to the problem, i.e. to express an N-terminally extended precursor of the desired polypeptide.
• the precursor molecule can thus be preferentially acylated in the desired lysine residue which in the case of GLP-1 is the lysine in position 26. After acylation the acylated precursor molecule is cleaved by suitable chemical or enzymatic means as described above and the desired acylated polypeptide can be isolated.
• the acylation step (iii) may be conducted at a pH between 7 and 12.
• the pH will be between 8 and 11.5 or between 9.0 and 10.5 and a pH value of about 9.5 to 10.5 has proven to be efficient.
• the temperature will be between minus 5 and 35°C and will typically be between 0 and 20°C or between 15 and 30°C.
• preferential acylating is meant to include and acylation process where acylation takes place at one or more preferred positions in the molecule in a higher degree than at other positions in the same molecule.
• the acylation at the preferred positions is preferably at least 50, more preferred at least 80 and most preferred 90-100% of the total acylation.
• N-terminal extension is meant a polypeptide sequence removably attached to the N-terminal amino acid residue in the desired polypeptide.
• the N-terminal extension may be 1-15 amino acid residues in length and will not comprise a Lys residue.
• the N-terminal extension will protect the expressed fusion polypeptide against proteolytic degradation within the host cell as described above.
• N-terminal extension includes the cleavage site for cleavage of the N-terminal extension from the desired polypeptide's N- terminal end. It will be understood that whenever a specific N-terminal extension or a sequence being comprised in the N-terminal extension is shown, then the given C-terminal amino acid residue will be the cleavage site directly linked to the N-terminal amino acid residue of the desired polypeptide.
• GLP-1 An example of a desired polypeptide is GLP-1.
• the amino acid sequence of GLP-1 is given i.a. by Schmidt et al. (Diabetologia 28 704-707 (1985). Although the interesting pharmacological properties of GLP-1 (7-37) and analogues thereof have attracted much attention in recent years only little is known about the structure of these molecules.
• the secondary structure of GLP-1 in micelles has been described by Thorton et al. ⁇ Biochemistry 33 3532- 3539 (1994)), but in normal solution,GLP-1 is considered a very flexible molecule.
• Gly 8 GLP-1 (7-37) designates a fragment of GLP-1 derived from GLP-1 (1-37) by deleting the amino acid residues Nos. 1 to 6 and substituting the naturally occurring amino acid residue in position 8 (Ala) by Gly.
• Lys 26 (N ⁇ -tetradecanoyl)-GLP-1 (7-37) designates GLP-1 (7-37) wherein the ⁇ -amino group of the Lys residue in position 26 has been tetradecanoylated.
• GLP-2 and glucagon both belonging to the GRF (growth hormone releasing factor) family of peptides having a His or Tyr in the N-terminal position and Ser, Ala or Gly in the next position, vide Adelhorts K. et al., The Journal of Biological Chemistry (1994) p 6275 -6278).
• POT is the Schizosaccharomyces pombe triose phosphate isomerase gene
• TPI1 is the S. cerevisiae triose phosphate isomerase gene
• Fibrillation is meant a process where so called “fibrils” are formed.
• Fi- brils is a well recognized and described phenomenon and may be composed of antiparallel ⁇ -sheets. Molecules like GLP's with little ⁇ -helical structure and a very flexible and little tertiary structure are very prone to aggregation that leads to precipitation and loss of yield if very crude chemical conditions are not taken in use such as alkaline treatment at pH -12.
• leader an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.
• signal peptide is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein.
• the function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum.
• the signal peptide is normally cleaved off in the course of this process.
• the signal peptide may be heterologous or homologous to the yeast organism producing the protein.
• a number of signal peptides may be used with the DNA construct of the invention including the YPS1 signal peptide (formally called the YAP3 signal peptide) or any functional analogue thereof (Egel-Mitani et al.
• pro-peptide means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell).
• the pro-peptide may be the yeast ⁇ -factor pro-peptide, vide US 4,546,082 and 4,870,008.
• the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature.
• Suitable synthetic pro-peptides are those disclosed in US 5,395,922; 5,795,746; 5,162,498 and WO 98/32867.
• the pro- peptide will preferably contain an endopeptidase processing site at the C-terminal end, such as a Lys- Arg sequence or any functional analog thereof.
• the polynucleotide sequence of the invention may be prepared synthetically by estab- lished standard methods, e.g. the phosphoamidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3:801-805.
• oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct.
• a currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR).
• the polynucleotide sequence of the invention may also be of mixed genomic, cDNA, and synthetic origin.
• a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the precursor molecule of the invention, after which the DNA sequence may be modified at a site by inserting synthetic oli- gonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides.
• the invention encompasses a vector which is capable of replicating in the selected microorganism or host cell and which carries a polynucleotide sequence encoding the precur- sor molecule of the invention.
• the recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
• the vector may contain any means for assuring self-replication.
• the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
• a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
• the vector may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
• the recombinant expression vector is capable of replicating in yeast. Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 ⁇ m replication genes REP 1-3 and origin of replication.
• the vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells.
• a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
• Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
• Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyl- transferase), pyrG (orotidine-5'-phosphate decarboxylase) and trpC (anthranilate synthase.
• Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
• a preferred selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125-130).
• the polynucleotide sequence is operably connected to a suitable promoter sequence.
• the promoter may be any nucleic acid sequence which shows transcrip- tional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intra-cellular polypeptides either homologous or heterologous to the host cell.
• suitable promoters for directing the transcription in a bacterial host cell are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha- amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus licheniformis penicillinase gene (penP).
• dagA Streptomyces coelicolor agarase gene
• sacB Bacillus subtilis levansucrase gene
• amyL Bacillus stearothermophilus maltogenic amylase gene
• amyQ Bacillus amyloliquefaciens alpha-amylase gene
• penP Bacillus lichen
• promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase.
• useful promoters are the Sac- charomyces cerevisiae MF ⁇ 1 , TPI, ADHm Gal or PGK promoters.
• the polynucleotide construct of the invention will also typically be operably connected to a suitable terminator.
• a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1 :419-434) or the CYC1 terminator.
• TPI terminator Alber et al. (1982) J. Mol. Appl. Genet. 1 :419-4314
• CYC1 terminator the procedures used to ligate the polynucleotide sequence of the invention, the promoter and the terminator, respectively, and to insert them into a suitable vector containing the information necessary for replication in the selected host, are well known to persons skilled in the art.
• the vector may be constructed either by first pre- paring a DNA construct containing the entire DNA sequence encoding the precursor molecule of the invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements followed by ligation.
• the present invention also relates to recombinant host cells, comprising a polynucleo- tide sequence encoding the precursor molecule of the invention.
• a vector comprising such polynucleotide sequence is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
• the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
• the host cell may be a pro- karyote or a eukaryote cell.
• Useful prokaryotes are bacterial cells such as gram positive bacteria including Bacillus and Streptomyces cells, or gram negative bacteria such as E. coli and Pseudomonas sp. Cells.
• Eukaryote cells may be mammalian, insect, plant, or fungal cells.
• the host cell is a yeast cell.
• the yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the precursor molecule.
• yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geot chum sp., and Geotrichum fermentans.
• the transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se.
• the medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms.
• the secreted precursor of the invention may then be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the precursor by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
• a salt e.g. ammonium sulphate
• an analogue is used to designate a peptide wherein one or more amino acid residues of the parent peptide have been substituted by another amino acid residue and/or wherein one or more amino acid residues of the parent peptide have been deleted and/or wherein one or more amino acid residues have been added to the parent peptide.
• Such addition can take place either at the N-terminal end or at the C- terminal end of the parent peptide or both.
• derivative is used in the present text to designate a peptide in which one or more of the amino acid residues of the parent peptide have been chemically modified, e.g. by alkylation, acylation, ester formation or amide formation.
• Fig. 1 shows the plasmid pKV304 which contain DNA encoding Arg 34 GLP-1 (1-37) under regulatory control of the TPI promoter and -terminator and the MFalpha prepro sequence.
• This plasmid is the starting plasmid for making expression plasmids for the precur- sor molecules according to the present invention.
• the host strain ME1719 is a diploid strain and has a phenotype which lacks two as- partyl protease activities, i.e. YPS1 (previously called YAP3) which cleaves C-terminal side of mono- or dibasic amino acid residues (Egel-Mitani, et al., YEAST 6: 127-137, 1990) and PEP4 a vacuolar protease A responsible for activation of other proteases such as protease B, carboxypeptidase Y, aminopeptidase I, RNase, alkaline phosphatase, acid threhalase and exopolyphosphatase.
• YPS1 previously called YAP3
• PEP4 a vacuolar protease A responsible for activation of other proteases such as protease B, carboxypeptidase Y, aminopeptidase I, RNase, alkaline phosphatase, acid threhalase and exo
• triose phosphate isomerase gene has been dis- rupted which phenotype makes it possible to utilize glucose in transformants grown on glucose containing medium.
• the genetic background of ME1719 is MATa/ ⁇ ⁇ yps1 ::ura3/ ⁇ yps1 ::URA3 pep4-3/pep4-3 ⁇ tpi::LEU2/ ⁇ tpi::LEU2 Ieu2/leu2 ⁇ ura3/ ⁇ ura3.
• Expression plasmids containing the N-terminally extended Arg 34 GLP-1 (7 . 37) were made as follows: Plasmid pKV304 containing DNA encoding Arg 34 GLP-1 (7 . 37) without an N- terminal extension was digested with either Eagl+Ncol or Eagl+Asp718. After agarose electrophoresis and GeneCleanTM III purification, fragments of 1.4 kb and 10 kb, respectively were isolated. Oligonucleotide adaptors corresponding to various N-terminal extensions of Arg GLP-1 (7 . 37) containing Ncol and Asp718 cleavage sites were likewise purified as described above.
• the 1.4 kb fragment (Eagl+Ncol), 10 kb fragment (Eagl+Asp718) and the adaptor fragment designed for the N-terminal extension of Arg 34 GLP-1 (7 ⁇ 7) (Ncol+Asp718) were ligated and transformed in E. coli strain MT172 and plasmid DNA was sequenced to verify the correct N-terminally extended Arg 34 GLP-1 (7 . 37) .
• Plasmid DNA was then transformed into yeast strain ME1719 and yeast transformants were isolated twice on MUPD selective plates. Yeast cells were cultured in 5 ml MUPD medium for 3 days at 30°C and culture supernatants were analyzed by HPLC and MALDI-MS (Matrix Assisted Laser Desorption/lnonisation Mass Spectrometry).
• Table 1 shows the different GLP-1 precursors and the yield compared to a control with no N-terminal extension.
• Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2)-GLP-1 was expressed in yeast and purified as previously described. Afterwards the peptide was acylated by use of N ⁇ -palmitoyl-Glu- ⁇ - succinimidyl- ⁇ -tert-butyl ester and deprotected by use of TFA. Reverse-phase HPLC-MS identified the desired product, Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2)-NN2211 acylated in position Lys-26 in a yield of 80 %. 20% of the acylation mixture was acylated both in Lys-26 and N-terminally. Since cleavage of both these peptides at Asn-His should yield the same product Arg 34 GLP-1 (7 . 37) it was decided to do the exploratory studies with the 80/20 mixture.
• FXa Glu-Glu-lle-Glu-Gly-Arg(SEQ ID NO: 16)- Arg 34 GLP-1 (7-37) was expressed and recovered as previously described. 1 mg lyophilized product was dissolved in 10 ml 0,02 M Tris and 2 mM CaCI 2 , pH 7,5. At time zero 40 units of FXa (Amersham Pharmacia Biotech, Product number: 27-0849-01) was added and the incubation was stopped after 2 hours at 30°C in a water bath by dilution 1 :1 with 1 M HAc. Determination by HPLC and MALDI-TOF showed 82 % correctly cleaved Arg 34 GLP-1 (7-37) and 18 % non-cleaved product Glu-Glu-lle-Glu-Gly-
• Arg(SEQ ID NO:16)- Arg 34 GLP-1 (7-3 7) - Arg 34 GLP-1 (7-37) can then be acylated by well established known methods.
• Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3)- Arg 34 GLP-1 (7-37) was expressed and recovered as previously described.
• 0,37 mg lyophilized product was dissolved in 900 ⁇ l 0,1 M NaAc and 5 mM CaCI 2 , pH 6,0.
• 100 ⁇ l of soluble C-terminal truncated kexin was added and the mixture was incubated in a water bath at 30°C. After 1 hour the reaction was stopped by addition of 2 M HAc 1 :1 and cleavage was determined by HPLC and MALDI-MS.
• Arg 34 GLP-1 (7-37) 66.9% was cleaved to Arg 34 GLP-1 (7-37) and the rest was uncleaved Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3)- Arg 34 GLP-1 (7-37) .
• Arg 34 GLP-1 (7-37) can then be acylated by well established known methods.
• Glu-Glu-Ala-Glu-Pro(SEQ ID NO:13 )-Arg 3 GLP-1 (7-37) was expressed and recovered as pre- viously described. 11 ,4 mg lyophilized product was dissolved in 50 ml 0,02 M Tris, HCI, pH 7,5. At time zero 500 ⁇ l was added of prolylendopeptidase (Sphingomonas capsulata prolylendopeptidase expressed in E.coli according to Kanatani et al, Archives of Biochemistry and Biophysics, 358, 141-148 (1998). Cleavage and formation of Arg 34 GLP-1 (7-37) are detected after 30 min incubation in a water bath at 30°C. Arg 34 GLP-1 (7-37) can now be acylated by well established, known methods.

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