WO2002079251A2 - Methode de fabrication de precurseurs d'insuline humaine - Google Patents

Methode de fabrication de precurseurs d'insuline humaine Download PDF

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WO2002079251A2
WO2002079251A2 PCT/DK2002/000191 DK0200191W WO02079251A2 WO 2002079251 A2 WO2002079251 A2 WO 2002079251A2 DK 0200191 W DK0200191 W DK 0200191W WO 02079251 A2 WO02079251 A2 WO 02079251A2
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insulin
insulin precursor
peptide
host cell
precursor
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PCT/DK2002/000191
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WO2002079251A3 (fr
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Thomas Børglum KJELDSEN
Svend Ludvigsen
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Novo Nordisk A/S
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    • 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
    • C07K14/62—Insulins

Definitions

  • 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.
  • 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 ⁇ -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.
  • 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 contain at least one Gly and will in general not be of more than 6 amino acid residues and will preferably not be of more than 4 amino acid residues in length.
  • the connecting peptide will contain 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.
  • 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.
  • the connecting peptide will not contain two adjacent basic amino acid residues (Lys.Arg).
  • cleavage from the A-chain will preferably be accomplished at a single Lys or Arg located at the N-terminal end of the A-chain.
  • the present invention is related to insulin precursors comprising a sequence of formula:
  • X ⁇ is up to 5 amino acid residues in length and Y is a cleavage site.
  • X- is of 1-4, 1-3 or 1-2 amino acid residues in length.
  • Y is Lys or Arg.
  • X 1 is GluGly; GluGluGl ; SerGly; AsnGly, ThrGly, AspGly; MetGly; AlaGly or HisGly.
  • sequence X Y can be (a) Glu- Glu-Gly-Lys(SEQ ID NO:1 , (b) Glu-Gly-Lys, (c) Ser-Gly-Lys, (d) Asn-Gly-Lys, (e).Thr-Gly- Lys, (f) Asp-Gly-Lys, (g) Met-Gly-Lys, (h) Ala-Gly-Lys, or (i) His-Gly-Lys.
  • the present invention is also related to polynucleotide sequences which code for the claimed insulin precursors.
  • the present invention is related to vectors containing such polynucleotide sequences and to host cells containing such polynucleotide sequences or vectors.
  • the invention in another aspect, relates to a process for producing insulin 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.
  • 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 desB30 human insulin or human insulin by in vitro enzymatic conversion.
  • the invention relates to a process for producing an acylated 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, (iii) converting the insulin precursor into desB30 human insulin and (iv) converting the desB30 human insulin into an acylated derivate by use of a convenient acylation method.
  • 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.
  • Fig. 1 represents the pAK855 S. cerevisiae expression plasmid expressing the TA57 leader-EEGEPK(SEQ ID NO:2)-B(1-29)-AlaAlaLys-A(1-21) precursor
  • Fig. 2. represents the nucleotide sequence of the expression cassette of the pAK855 yeast expression plasmid and the inferred amino acid sequence (SEQ ID NO: 3 and 4).
  • 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.
  • IP is meant a single-chain insulin precursor in which a desB30 chain is linked to the A chain of insulin via a connecting peptide. The single-chain insulin precursor will contain correctly positioned disulphide bridges (three) as in human insulin.
  • 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.
  • 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 desB30 human insulin.
  • 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.
  • significantly increased production 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.
  • immediate N-terminal to is meant to illustrate the situation where an amino acid residue or a peptide sequence is directly linked at its C-terminal end to the N- terminal end of another amino acid residue or amino acid sequence by means of a peptide bond.
  • POT is the Schizosaccharomyces pombe triose phosphate isomerase gene
  • TPI1 is the S. cerevisiae triose phosphate isomerase gene
  • 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 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.
  • 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.
  • 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 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.
  • oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and Hgated 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 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.
  • 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 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.
  • the recombinant expression vector is capable of replicating in yeast organisms.
  • 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 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 TP ⁇ 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 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.
  • 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 Saccharomyces cerevisiae Ma1 , TPI, ADH 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).
  • TPI terminator Alber et al. (1982) J. Mol. Appl. Genet. 1:419-434.
  • 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.
  • 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.
  • 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 per se.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms.
  • the secreted insulin precursor of the invention 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.
  • 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.
  • proteolytic enzymes are trypsin or Achromobacter lyticus protease .
  • 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.
  • 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.
  • POT Schizosaccharomyces pombe triose phosphate isomerase gene
  • 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-Xfoal fragment encoding the fusion of the leader and insulin precursor.
  • EcoRI-Xbal fragment of pKFN1003 is simply replaced by an EcoRI-X al fragment encoding the leader insulin precursor of interest.
  • EcoRI- bal 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/MAT pep4-3/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.
  • YPGaL 1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate
  • 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 Na 2 EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg Novozym®234.
  • Synthetic genes encoding fusion proteins consisting of the insulin precursor associated with a leader sequence consisting of a pre-peptide (signal peptide) and a pro-peptide were constructed using PCR under standard conditions (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) and E.H.F. polymerase (Boehringer Mannheim GmbH, Sandhoefer Strasse 116, Mannheim, Germany). The resulting DNA fragments were isolated and digested with endonucleases and purified using the Gene Clean kit (Bio101 Inc., La Jolla, CA, USA). Standard methods were used for DNA ligation and transformation of E. coli cells were performed by the CaCI 2 method (Sambrook et al. (1989) supra).
  • Plasmids were purified from transformed E. coli cells using QIAGEN columns (QIAGEN, Hilden, Germany). Nucleotide sequences were determined using the ALF Pharmacia Biotech DNA sequencing system with purified double-stranded plasmid DNA as template. Oligonucleotide primers for PCR were obtained from DNA technology (Arhus, Denmark).
  • the pAK855 S. cerevisiae expression plasmid expressing the TA57 leader-EEGEDK(SEQ ID NO:2)-insulin precursor fusion protein was constructed based on the S. cerevisiae-E. coli shuttle POT plasmid (U.S. patent 5,871 ,957).
  • L-IP indicates the fusion protein expression cassette encoding the leader-insulin precursor fusion protein
  • TPI-PROMOTER is the S. cerevisiae TPI1 promoter
  • TPI-TERMINATOR is the S. cerevisiae TPI1 terminator
  • TPI-POMBE indicates the S. pombe POT gene used for selection in S.
  • ORIGIN indicates a S. cerevisiae origin of replication derived from the 2 ⁇ m plasmid
  • AMP-R indicates the ⁇ -lactamase gene conferring resistance toward ampicillin, facilitating selection in E. coli
  • OR1GIN-PBR322 indicates an E. co// origin of replication.
  • DNA encoding a number of fusions proteins of leader sequences and insulin precursors with different mini C-peptides was generated by PCR using appropriate oligonucleotides as primers, as described below. Standard methods were used to subclone DNA fragments encoding the leader-insulin precursor fusion proteins into the CPOT expression vector in the following configuration: leader-Lys-Arg-spacer-insulin precursor, where Lys-Arg is a potential dibasic endoprotease processing site and spacer is an N- terminal extension.
  • EEGEPK (SEQ ID NO:2) was inserted between the DNA encoding the leader and the insulin precursor (Kjeldsen et al. (1999b.) J. Biotechnology, 75 195-208). However, the present of the spacer peptide is not mandatory.
  • the insulin precursor was secreted as a single-chain N-terminally extended insulin precursor with a mini C-peptide, connecting Lys B29 and Gi ⁇ 1 . After purification of the insulin precursor and proteolytic removal of the N-terminal extension and the mini C-peptide, the amino acid Thr B3 ° can be added to Lys B29 by enzyme-mediated transpeptidation, to generate human insulin (Markussen, et al. (1987) in "Peptides 1986" (Theodoropoulos, D., Ed.), pp. 189-194, Walter de Gruyter & Co., Berlin.).
  • the synthetic mini C-peptides feature typically an enzymatic processing site (Lys) at the C-terminus which allows enzymatic removal of the synthetic mini C-peptide. Randomization was performed using doped oligonucleotides which introduced codon(s) variations at one or more positions of the synthetic mini C-peptides. Typically one of the two primers (oligonucleotides) used for PCR was doped. Examples of primers are: Primer A; 5'-TTGCTTAAATCTATAACTAC-3' (SEQ ID NO: 5) Primer B:
  • PCR was typically performed as indicated below: 5 ⁇ l Primer A (20 pmol), 5 ⁇ l Primer B (20 pmol), 10 ⁇ l 10X PCR buffer, 8 ⁇ l dNTP mix, 0.75 ⁇ l E.H.F. enzyme, 1 ⁇ l pAK885 plasmid as template (approximately 0.2 ⁇ g DNA), and 70.25 ⁇ l distilled water.
  • one cycle typically was 95° C for 45 sec; 55° C for 1 min; 72° C for 1.5 min.
  • the PCR mixture was subsequently loaded onto an 2 % agarose gel and electrophoresis was performed using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated by the Gene Clean kit.
  • Fig. 2 shows the nucleotide sequence of the pAK855 DNA expression cassette used as template for PCR and inferred amino acids of the encoded fusion protein (TA57-leader- EEGEPK(SEQ ID NO:2)-insulin precursor of pAK855 (SEQ ID NO:3 and 4).
  • the purified PCR DNA fragment was dissolved in water and restriction endonucleases buffer and digested with suitable restriction endonucleases (e.g. Bgl II and Xba I) according to standard techniques.
  • suitable restriction endonucleases e.g. Bgl II and Xba I
  • the Bglll-Xbal DNA fragments were subjected to agarose electrophoresis and purified using The Gene Clean Kit.
  • the digested and isolated DNA fragments were ligated together with a suitable vector (e.g. of the CPOT type) using T4 DNA ligase and standard conditions.
  • the ligation mix was subsequently transformed into a competent E. coli strain (R-, M+) followed by selection with ampicillin resistance. Plasmids from the resulting E. coli's were isolated using QIAGEN columns.
  • the plasmids were subsequently used for transformation of a suitable S. cerevisiae strainMT663 (MATa/MAT pep4-3/pep4-3 HlS4/his4 tpi::LEU2/tpi::LEU2 Cir + ).
  • Individual transformed S. cerevisiae clones were grown in liquid culture, and the quantity of secreted insulin precursor the culture supematants was determined by RP-HPLC.
  • the DNA sequence encoding the synthetic mini C-peptide of the expression plasmids from S. cerevisiae clones secreting increased quantity of the insulin precursor were then determined.
  • Table 1 shows the insulin precursors generated by the above method and production yield expressed as a percent of control. Fermentation was conducted at 30°C for 72 h in 5 ml YPD. Yield of the insulin precursor was determined by RP-HPLC of the culture supernatant, and is expressed relative to the yield of a control strain expressing a leader-insulin precursor fusion protein in which the B29 residue is linked to the A1 residue by a mini C-peptide Ala-Ala- Lys.
  • YAP3 is the YAP3 signal sequence.
  • sequence EEGEPK (SEQ ID NO:2) is an N- terminal extension to the B-chain and TA57 is a synthetic pro-sequence QPIDDTESQTTSVNLMADDTESAFATQTNSGGLDWGLISMAKR (SEQ ID NO:7).
  • insulin precursors with a Gly in the mini C-peptide under the control of the synthetic leader TA37 QPIDDTESNTTSVNLMADDTESRFATNTTLAGGLDWNLI-SMAKR (SEQ ID NO:8) and the YAP3 signal sequence were expressed in the yeast host MT663.
  • the precursors all comprised an N-terminal extension EEGEPK (SEQ ID NO:2).
  • the increase in expression yield compared to a control is shown in Table 2.
  • insulin precursors with a Gly in the mini C-peptide under the control of the ⁇ -factor leader sequence were expressed in the yeast host MT663.
  • the insulin precursors were expressed with and without an N- terminal extension EEAEAEAPK (SEQ ID NO: 12).
  • the increase in expression yield compared to a control is shown in Table 3.

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Abstract

L'invention concerne de nouveaux précurseurs d'insuline comprenant un peptide C (mini-peptide C) qui contient jusqu'à 6 résidus d'aminoacides, et au moins un Gly. On peut convertir les précurseurs en insuline humaine ou en insuline humaine desB30.
PCT/DK2002/000191 2001-04-02 2002-03-21 Methode de fabrication de precurseurs d'insuline humaine WO2002079251A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004085472A1 (fr) * 2003-03-27 2004-10-07 Novo Nordisk A/S Procede de fabrication de precurseurs de l'insuline humaine et insuline humaine
US6924120B1 (en) * 1998-03-31 2005-08-02 Tonghua Gantech Biotechnology, Ltd. Chaperone protein containing an intramolecular chaperone-like sequence and its application to insulin production
WO2014195452A1 (fr) 2013-06-07 2014-12-11 Novo Nordisk A/S Procédé de fabrication de polypeptides d'insuline mature
CN106749616A (zh) * 2016-12-19 2017-05-31 昂德生物药业有限公司 B30位苏氨酸缺失人胰岛素晶体的制备方法
WO2018047062A1 (fr) * 2016-09-06 2018-03-15 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Dérivés de pro-insuline

Citations (5)

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WO2014195452A1 (fr) 2013-06-07 2014-12-11 Novo Nordisk A/S Procédé de fabrication de polypeptides d'insuline mature
JP2016521701A (ja) * 2013-06-07 2016-07-25 ノヴォ ノルディスク アー／エス 成熟インスリンポリペプチドを作製するための方法
WO2018047062A1 (fr) * 2016-09-06 2018-03-15 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Dérivés de pro-insuline
AU2017322552B2 (en) * 2016-09-06 2021-12-02 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Proinsulin derivatives
US11230585B2 (en) 2016-09-06 2022-01-25 Chemical & Biopharmaceutical Laboratories Of Patra Proinsulin derivatives
CN106749616A (zh) * 2016-12-19 2017-05-31 昂德生物药业有限公司 B30位苏氨酸缺失人胰岛素晶体的制备方法
CN106749616B (zh) * 2016-12-19 2021-09-03 华润昂德生物药业有限公司 B30位苏氨酸缺失人胰岛素晶体的制备方法

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