WO2004085472A1

WO2004085472A1 - Method for making human insulin precursors and human insulin

Info

Publication number: WO2004085472A1
Application number: PCT/DK2004/000209
Authority: WO
Inventors: Thomas Børglum KJELDSEN
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2003-03-27
Filing date: 2004-03-26
Publication date: 2004-10-07
Anticipated expiration: 2005-09-27

Abstract

Novel insulin precursors comprising a connecting peptide (mini C-peptide) of up to 4 amino acid residues and comprising a sequence Glu-Asp. Glu-Glu, Asp-Asp, or Asp-Glu are prepared in high yield in transformed microorganisms, such as yeast. The precursors can be converted into human insulin or desB30 human insulin.

Description

METHOD FOR MAKING HUMAN INSULIN PRECURSORS AND HUMAN INSULIN

BACKGROUND Yeast organisms produce a number of proteins that have a function outside the cell.

Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852). Insulin is a polypeptide hormone secreted by β-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.

The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids, in the configuration: prepeptide - B - Arg Arg - C - Lys Arg -A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.

Three major methods have been used for the production of human insulin in microorganisms. Two involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7^th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, IL. pp 729-739), or use of a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a limited number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide US 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

SU ARY OF THE INVEMTIOM The present invention features novel connecting peptides (C-peptides) which confer an increased production yield of insulin precursor molecules when expressed in a transformed microorganism, in particular yeast. Such insulin precursors can then be converted into human insulin, desB30 human insulin or certain acylated human insulins by one or more suitable, well known conversion steps

The connecting peptides of the present invention consist of 3-4 amino acid residues and contain at least one Asp or one Glu.

The connecting peptide contains a cleavage site at its C-terminal end enabling in vitro cleavage of the connecting peptide from the A chain. Such cleavage site may be any convenient cleavage site known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases; Acromobactor lyticus protease or by a carboxypeptidase protease. The cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.

Cleavage of the connecting peptide from the B chain is enabled by cleavage at the natural Lys^B29 amino acid residue in the B chain giving rise to a desB30 insulin precursor. If the insulin precursor is to be converted into human insulin, the B30 Thr amino acid residue can then be added in vitro by well known, enzymatic procedures. The desB30 insulin may also be converted into an acylated insulin analogue as disclosed in US 5,750,497 and US 5,905,140.

In a more specific aspect, the present invention is related to insulin precursors or insulin analogue precursors comprising a sequence of formula: B(1 -29) - X_rX₂-X3- Y - A(1 -21 ), wherein X^ is any amino acid except Cys or is a peptide bond, X₂ is Asp or Glu, X₃ is Asp or Glu and Y is a cleavage site; B(1-29) is the human B-chain or an analogue thereof lacking the amino acid in position B(30) and A(1-21) is the human insulin A chain or an analogue thereof.

In one embodiment X-i is a peptide bond, X₂ is Glu or Asp, X₃ is Glu or Asp and Y is Lys or Arg. Thus the sequence X₂-X₃-Y can be (a) Glu-Asp-Lys, (b) Glu-Glu-Lys, (c) Asp- Asp-Lys, (d) Asp-Glu-Lys, Glu-Asp-Arg, (e) Glu-Glu-Arg, (f) Asp-Asp-Arg, or (f) Asp-Glu-Arg. The present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin analogue precursors. In a further aspect the present invention is related to vectors containing such polynucleotide sequences and to host cells containing such polynucleotide sequences or vectors.

In another aspect, the invention relates to a process for producing insulin precursors or insulin analogue precursors in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors of the invention under suitable conditions for expression of said precursor and (ii) isolating the insulin precursor from the culture medium.

In still a further aspect, the invention relates to a process for producing human insulin or desB30 human insulin comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor of the invention; (ii) isolating the insulin precursor from the culture medium and (iii) converting the insulin precursor into desBSO human insulin or human insulin by in vitro enzymatic conversion.

In still a further aspect, the invention relates to a process for producing an acylated desBSO human insulin comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor of the invention; (ii) isolating the insulin precursor from the culture medium, (iii) converting the insulin precursor into desBSO human insulin and (iv) converting the desBSO human insulin into an acylated derivate by use of a convenient acylation method.

.In a further aspect, the present invention is related to a process for making human insulin or a human insulin analogue, said method comprising converting an insulin precursor or insulin analogue precursor according as described above into human insulin or a human insulin analogue by suitable in vitro chemical or enzymatic conversion. In one embodiment of the present invention, the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from to the genus Saccharomyces. In a further embodiment, the yeast host cell is selected from the species Saccharomyces cerevisiae.

DETAILED DESCRIPTION Abbreviations and nomenclature.

By "connecting peptide" or "C-peptide" is meant the connection moiety "C" of the B-C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the A chain. A "mini C-peptide" or "connecting peptide" such as those described herein, connect B29 to A1 and differ in sequence and length from that of the natural C-peptide.

With "desB30" or "B(1-29)" is meant a natural insulin B chain lacking the B30 amino acid residue, "A(1-21)" means the natural insulin A chain, The mini C-peptide and its amino acid sequence is indicated in the three letter amino acid code.

By "insulin precursor" is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into human insulin or desBSO human insulin.

By "insulin analogue precursor" is meant an insulin precursor molecule having one or more mutations, substitutions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule. The insulin analogues are preferably such wherein one or more of the naturally occurring amino acid residues, preferably one, two, or three of them, have been substituted by another codable amino acid residue. In one embodiment, the instant invention comprises analogue molecules having position 28 of the B chain altered relative to the natural human insulin molecule. In this embodiment, position 28 is modified from the natural Pro residue to one of Asp, Lys, or lie. In a preferred embodiment, the natural Pro residue at position B28 is modified to an Asp residue. In another embodiment Lys at position B29 is modified to Pro; Also, Asn at position A21 may be modified to Ala, Gin, Glu, Gly, His, lie, Leu, Met, Ser, Thr, Trp, Tyr orVal, in particular to Gly, Ala, Ser, orThr and preferably to Gly. Furthermore, Asn at position B3 may be modified to Lys. Further non- limiting examples of insulin analogue precursors are des(B30) human insulin, insulin analogues wherein Phe^B1 has been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B1. The present invention features novel mini C-peptides connecting position 29 of the insulin B chain and position 1 of the insulin A chain which significantly increased production yields in a yeast host cell. By the term "significantly increased production," "increased fermentation yield," and the like, is meant an increase in secreted amount of the insulin precursor molecule present in the culture supernatant compared to the yield of an insulin precursor with no Gly in the mini C peptide. An "increased" fermentation yield is an absolute number larger than the control; preferably, the increase is 50% or more larger than the control.

"POT' is the Schizosaccharomyces pombe triose phosphate isomerase gene, and "TPI1" is the S. cerevisiae triose phosphate isomerase gene. By a "leader" is meant an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.

The term "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 which may be used with the DNA construct of the invention including yeast aspartic protease 3 (YAP3) signal peptide or any functional analog (Egel-Mitani et al. (1990) YEAST 6:127-137 and US 5,726,038) and the α-factor signal of the MFo gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae. Strathern et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NY and US 4,870,00. The term "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 α-factor pro-peptide, vide US 4,546,082 and 4,870,008. Alternatively, 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 endopeplidase 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 established 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. According to the phosphoamidite method, 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. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides 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 cell line and which carries a polynucleotide sequence encoding the insulin precursors 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. Alternatively, 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. Furthermore, 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 vectors 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.

In a preferred embodiment, the recombinant expression vector is capable of replicating in yeast organisms. Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 μm 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 ortetracycline resistance. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (omithine carbamoyltransferase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase) 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). In the vector, the polynucleotide sequence is operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of 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). Examples of suitable 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. In a yeast host, useful promoters are the Saccharomyces cerevisiae Ma1, TPI, ADH or PGK promoters.

The polynucleotide construct of the invention will also typically be operably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1: 19-434). The procedures used to ligate the polynucleotide sequence of the invention, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication in the selected host cells, are well known to persons skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence 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 (such as the signal, pro-peptide, mini C-peptide, A and B chains) followed by ligation.

The present invention also relates to recombinant host cells, comprising a polynucleotide sequence encoding the insulin precursors 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 unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp. Eukaryote cells may be mammalian, insect, plant, or fungal cells. In a preferred embodiment, 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 insulin precursor and insulin precursor analogs of the invention. Examples of suitable 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, Geotrichum 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 perse. The medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms. The secreted insulin precursor of the invention, a significant proportion of which will be present in the medium in correctly processed form, may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the insulin 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.

The insulin precursors of the invention may be expressed with an N-terminal amino acid residue extension, as described in U.S. Patent No. 5,395,922, and European Patent No. 765.395A, both of which patents are herein specifically incorporated by reference. The extension is found to be stably attached to the insulin precursor of the invention during fermentation, protecting the N-terminal end of the insulin precursor against the proteolytic activity of yeast proteases such as DPAP. The presence of an N-terminal extension on the insulin precursor may also serve as a protection of the N-terminal amino group during chemical processing of the protein, i.e. it may serve as a substitute for a BOC (t-butyl- oxycarbonyl) or similar protecting group.

The N-terminal extension may be removed from the recovered insulin precursor by means of a proteolytic enzyme which is specific for a basic amino acid (e.g., Lys) so that the terminal extension is cleaved off at the Lys residue. Examples of such proteolytic enzymes are trypsin or Achromobacter lyticus protease. After secretion to the culture medium and recovery, the insulin precursor of the invention will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and the mini C-peptide to give desB30 insulin. DesB30 insulin may then be converted into human insulin by adding a Thr in position B30. Conversion of the insulin precursor into human insulin by a suitable enzymatic conversion by means of trypsin or an Achromobacter lyticus protease in the presence of an L-threonine ester followed by conversion of the threonine ester of the insulin into insulin by basic or acid hydrolysis is described in US patent specification No. 4,343,898 or 4,916,212 or Research Disclosure, September 1994/487 the disclosures of which are incorporated by reference hereinto. The desB30 insulin may also be converted into an acylated insulin derivative as disclosed in US 5,750,497 and US 5,905,140 the disclosures of which are incorporated by reference hereinto.

The present invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed. All references cited are herein specifically incorporated by reference for all that is described therein.

EXAMPLES General Procedures All expressions plasmids are of the C-POT type, similar to those described in EP 171

142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae. The plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-Xi al fragment encoding the fusion of the leader and insulin precursor. In order to express different fusion proteins, the EcoRI-Xfcal fragment of pKFN1003 or a similar C- POT type expreseeion plasmid is simply replaced by an EcoRI-Xfoal fragment encoding the leader insulin precursor of interest. Such EcoR\-Xba\ fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques. Yeast transformants were prepared by transformation of the host strain: S. cerevisiae strain MT663 (MATa/MATαpep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir⁺). The yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278. MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6. 100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM Na₂EDTA pH = 8.0 and 6.7 mg/ml dithiotreitol. The suspension was incubated at 30°C for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na₂EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg Novozym®234. The suspension was incubated at 30°C for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM CaCI₂, 10 mM Tris HCI (Tris = Tris(hydroxymethyl)aminomethane) pH = 7.5) and resuspended in 2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixed with approx. 0.1 mg of plasmid DNA and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCI₂, 10 mM Tris HCI, pH = 7.5) was added and the mixture left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCI₂) and incubated at 30°C for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory) containing 1.2 M sorbitol plus 2.5% agar) at 52°C was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium.

S. cerevisiae strain MT663 transformed with expression plasmids were grown in YPD for 72 h at 30°C. Quantitation of the insulin-precursor yield in the culture supernatants was performed by reverse-phase HPLC analysis with human insulin as an external standard (Snel & Damgaard (1988) Proinsulin heterogenity in pigs. Horm. Metabol. Res. 20:476-488). Table 1 shows the insulin precursors generated by the above method and production yield expressed as a percent of control. Table 1

Expression of a human single-chain desBSO insulin precursor

Claims

What we claim is:

1. An insulin precursor or an insulin analogue precursor comprising a sequence of formula:

B(1-29) - X1-X2-X3- Y - A(1-21), wherein X₁ is any amino acid except Cys or is a peptide bond, X₂ is Asp or Glu, X₃ is Asp or Glu, Y is a cleavage site, B(1-29) is the human B-chain or an analogue thereof lacking the amino acid in position B(30) and A(1-21) is the human insulin A chain or an analogue thereof.

2. An insulin precursor or an insulin analogue precursor according to claim 1, wherein X-i is a peptide bond and Y is Lys or Arg.

3. An insulin precursor or an insulin analogue precursor according to claim 2, wherein X₂ is Glu, X₃ is Asp, and Y is Lys or Arg.

4. An insulin precursor or an insulin analogue precursor according to claim 2, wherein X₂ is Asp, X₃ is Asp, and Y is Lys or Arg.

5. An insulin precursor or an insulin analogue precursor according to claim 2, wherein X₂ is Asp, X₃ is Glu, and Y is Lys or Arg.

6. An insulin precursor or an insulin analogue precursor according to claim 2, wherein X₂ is Glu, X₃ is Glu, and Y is Lys or Arg.

7. An insulin precursor or insulin analogue precursor according to claim 1 , wherein B(1-29) is the human B(1-29) chain and A(1-21) is the human insulin A chain.

8. An insulin precursor or an insulin analogue precursor according to claims 3-7, wherein Y is Lys.

9. A polynucleotide sequence encoding an insulin precursor or an insulin analogue precursor according to claim 1.

10. An expression vector comprising a polynucleotide of claim 9.

11. A host cell transformed with the vector claim 10.

12. A process for making an insulin precursor or an insulin analogue precursor said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin analogue precursor according to any of claims 1-8 under suitable culture conditions for expression of said insulin precursor or insulin analogue precursor ; and (ii) isolating the expressed insulin precursor or insulin analogue precursor.

13. A process according to claim 12, wherein the host cell is a yeast host cell

14. A process for making human insulin or a human insulin analogue, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin analogue precursor according to any of claims 1-8 under suitable culture conditions for expression of said insulin precursor or insulin analogue precursor ; (ii) isolating the expressed insulin precursor or insulin analogue precursor and (iii) converting the insulin precursor or insulin analogue precursor into human insulin or a human insulin analogue by suitable in vitro chemical or enzymatic conversion.

15. A process for making human insulin or desB30 human insulin, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursor according to any of claims 1-8 under suitable culture conditions for expression of said precursor; (ii) isolating the insulin precursor from the culture medium and (iii) converting the insulin precursor to human insulin or desBSO human insulin by suitable in vitro chemical or enzymatic conversion.

16. A process according to claim 14 or 15, wherein the host cell is a yeast host cell line.

17. A process for making human insulin or a human insulin analogue, said method comprising converting an insulin precursor or insulin analogue precursor according to claim 1 into human insulin or a human insulin analogue by suitable in vitro chemical or enzymatic conversion.