WO1995017510A1 - Procede de production d'un peptide i du type glucagon - Google Patents

Procede de production d'un peptide i du type glucagon Download PDF

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Publication number
WO1995017510A1
WO1995017510A1 PCT/DK1994/000487 DK9400487W WO9517510A1 WO 1995017510 A1 WO1995017510 A1 WO 1995017510A1 DK 9400487 W DK9400487 W DK 9400487W WO 9517510 A1 WO9517510 A1 WO 9517510A1
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glp
precursor
analogue
dna construct
construct encoding
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PCT/DK1994/000487
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English (en)
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Jesper Skou Rasmussen
Lars Thim
Søren Erik BJØRN
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Novo Nordisk A/S
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Priority to AU12725/95A priority Critical patent/AU1272595A/en
Publication of WO1995017510A1 publication Critical patent/WO1995017510A1/fr

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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
    • C07K14/605—Glucagons

Definitions

  • the present invention relates to a method of producing glucagon-like peptide or analogues or derivatives thereof.
  • Mammalian proglucagon has been demonstrated to contain three different though homologous petides, glucagon, glucagon-like peptide 1 (also termed GLP-1) and glucagon-like peptide 2 (cf. L.C. Lopez et al., Proc. Natl. Acad. Sci. USA 80, 1983, pp. 5485-5489, and G.I. Bell et al, Nature 302, 1983, pp. 716-718). Prior to 1985, no definite biological activity of GLP-1 had been reported.
  • GLP-l(l-36)amide like glucagon, stimulates insulin release from isolated precultured rat pancreatic islets in the presence of glucose in a dose-dependent manner (Schmidt, W.E. et al. Diabetologia 28 (1985) 704-7).
  • GIP glucose dependent insulinotropic peptide
  • Schmidt et al. suggested that an even stronger glucagon- and/or GIP-like biological activity could be expected with GLP-1(7- 36) than with the intact peptide.
  • GLP-1 fragments GLP-l(7-37) and GLP-l(7-36)amide and analogues and functional derivatives thereof are given in formula I:
  • the present invention relates to a method of producing glucagon-like peptide 1 (GLP-1) 7-36 or an analogue thereof in a bacterium, the method comprising
  • step (b) transforming a suitable bacterium with the expression vector prepared in step (a),
  • analogue is intended to indicate a peptide molecule which is functionally equivalent to GLP-1, i.e. which has substantially the same biological activity as native GLP-1, but whose amino acid sequence differs from that of native GLP-1 by one or more amino acids.
  • Analogues of GLP-1 may suitably be prepared by modifying the DNA sequence coding for native GLP-1 by substituting one or more nucleotides in the sequence, or by inserting one or more codons into the sequence, adding one or more codons at either end of the sequence, or deleting one or more codons at either end of or within the sequence. Examples of suitable GLP-1 analogues are described in, e.g. WO 91/11457.
  • expression cassette is intended to indicate the DNA construct containing two or more consecutive DNA sequences coding for GLP- 1(7-36) or analogue thereof preceded by a single promoter controlling the expression of the entire DNA construct.
  • all DNA sequences coding for GLP-l(7-36) or analogues thereof present in the construct are expressed from this one promoter, rather than from separate promoters preceding each DNA sequence. Consequently, the DNA construct is expressed as one amino acid sequence (the GLP-1 precursor) containing two or more copies of GLP-l(7-36) or an analogue thereof.
  • the host cell used in the process of the invention may be any suitable bacterium which, on cultivation, produces large amounts of the GLP-1 precursor.
  • suitable bacteria may be grampositive bacteria such as Bacillus subtilis. Bacillus licheniformis, Bacillus lentus. Bacillus brevis. Bacillus stearothermophilus. Bacillus alkalophilus. Bacillus amyloliquefaciens. Bacillus coagulans. Bacillus circulans. Bacillus lautus or Streptomyces lividans, or gramnegative bacteria such as Escherichia coli.lt has been found that E.coli is able to produce a high yield of the GLP-1 precursor and is therefore a preferred host organism. In E.coli is able to produce a high yield of the GLP-1 precursor and is therefore a preferred host organism. In E.coli is able to produce a high yield of the GLP-1 precursor and is therefore a preferred host organism. In E.coli is able to produce a high yield of the
  • the GLP-1 precursor is typically produced in the form of inclusion bodies.
  • the transformation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Ed., Cold Spring Harbor, NY, 1989).
  • the DNA construct encoding the GLP-1 precursor is preceded by a suitable promoter sequence, e.g. the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis ⁇ -amylase gene, Bacillus amyloliquefaciens BAN amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters, or the E. coli lac promoter.
  • a suitable promoter sequence e.g. the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis ⁇ -amylase gene, Bacillus amyloliquefaciens BAN amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambd
  • the DNA construct encoding the GLP-1 precursor may also be preceded by a ribosome binding site of of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis ⁇ -amylase gene, Bacillus amyloliquefaciens BAN amylase gene, Bacillus subtilis alcaline protease gene, Bacillus pumilus xylosidase gene, or E. coli lac gene.
  • the DNA construct of the invention comprising two or more consecutive DNA sequences encoding GLP- 1(7-36) may suitably be prepared by ligating two or more cDNA sequences encoding GLP- 1(7-36) which may, for instance, be obtained by preparing a mammalian, in particular human, cDNA library and screening for DNA sequences coding for GLP- 1(7-36) by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, 1982).
  • the DNA construct of the invention may also be produced by ligating two or more DNA sequences coding for GLP- 1(7-36) or an analogue thereof prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22. 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3. 1984, pp. 801-805.
  • oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, ligated or alternatively assembled by PCR overlap extension as described in R.M. Horton, Gene 77, 1989, pp. 61-68, and cloned in an appropriate vector.
  • the DNA construct encoding the GLP-1 precursor may advantageously comprise three or more, in particular four or more or six or more, consecutive DNA sequences coding for GLP- 1(7-36) or an analogue thereof.
  • each of the DNA sequences coding for GLP-l(7-36) or an analogue thereof may be synthesized in such a way that it contains a high proportion of alternative codons (i.e. different codons specifying the same amino acid) to minimize the homology between the GLP- 1(7-36) encoding sequences.
  • the expression vector comprising the DNA construct as described above may be any vector which is capable of replicating autonomously in a given host organism, typically a plasmid or bacteriophage.
  • the DNA sequence encoding the GLP-1 precursor should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host organism. Examples of suitable promoters are given above.
  • the vector may also comprise a selectable marker, e.g. a gene whose product confers antibiotic resistance, such as ampicillin, chloramphenicol or tetracyclin resistance, or the 5 da! genes from B.subtilis or B.licheniformis.
  • a selectable marker e.g. a gene whose product confers antibiotic resistance, such as ampicillin, chloramphenicol or tetracyclin resistance, or the 5 da! genes from B.subtilis or B.licheniformis.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing bacteria.
  • the GLP-1 precursor may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells to recover an 10 intracellular product, 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
  • the GLP-1 precursor recovered in step (d) of the process of the invention is 15 subsequently processed, in particular by enzymatic processing procedures known perse in the art.
  • the enzyme used for processing should preferably be one which is specific for arginine residues, such as trypsin, trypsin-like protease derived from Fusarium oxyspomm (WO 89/6270), clostripain (W.M. Mitchell et alsky Methods Enzvmol. 19. 1970, p. 635), mouse submaxillary gland protease (M. Levy et al., Methods Enzvmol. 19, 20 1970, p. 672), or thrombin or other proteolytic enzymes of the blood coagulation cascade.
  • the N-terminal amino acid residue of the GLP-1 precursor When expressed in E. coli, the N-terminal amino acid residue of the GLP-1 precursor will be a methionine. This implies that, after the GLP-1 precursor has been processed, a fraction of the recovered GLP- 1(7-36) will be preceded by Met. To obtain authentic 25 GLP-l(7-36), the N-terminal methionine will either have to be removed, e.g. by treatment with CNBr, or the fraction containing the Met-GLP- 1(7-36) will have to be discarded. To reduce the costs of production and/or to improve the yield of authentic GLP-l(7-36), the DNA construct encoding the GLP-1 precursor is preferably preceded by a presequence.
  • the presequence codes for a prepeptide which has an arginine as the C-terminal amino acid residue.
  • the prepeptide may suitably be cleaved off enzymatically, preferably with an arginine-specific protease, such as trypsin, trypsin- like protease derived from Fusarium ox sporum, clostripain, mouse submaxillary gland protease, or thrombin or other proteolytic enzymes of the blood coagulation cascade, so that it may be processed in the same step as the GLP-1 precursor.
  • the prepeptide may subsequently be separated from the resulting GLP- 1(7-36) by conventional procedures, such as chromatography, extraction and/or precipitation.
  • the DNA construct encoding the GLP-1 precursor may comprise a spacer sequence between two or more of the GLP-l(7-36) encoding sequences.
  • the spacer peptide encoded by the spacer sequence may comprise at least one, and preferably 2-5 amino acids.
  • the identity of the amino acids in the spacer peptide is not critical, but it is assumed that the presence of acid amino acids in the spacer peptide would be an advantage as this would tend to expose the peptide for processing.
  • the C-terminal amino acid of the spacer should preferably be arginine so that it may be cleaved off enzymatically by an arginine-specific enzyme as indicated above.
  • Advantages of including one or more spacer sequences might be that the GLP-1 precursor will tend to precipitate in the form of inclusion bodies so as to provide a better yield of the precursor, and that processing of the precursor to GLP- 1(7-36) may be optimized.
  • the DNA construct encoding the GLP-1 precursor may also be followed by a sequence coding for a post-peptide which, like the spacer peptide(s) serves the purpose of increasing the yield of the GLP-1 precursor, e.g. in the form of inclusion bodies.
  • the number of amino acids in the post-peptide may suitably be from 1 to 10.
  • the GLP-l(7-36) or analogue thereof resulting from the processing of the GLP-1 precursor in step (e) of the method of the invention may be used per se as an active component in a pharmaceutical composition.
  • the term "derivative" is intended to mean a peptidic compound resulting from any enzymatic or chemical modification of GLP-l(7-36) resulting in a compound with insulinotrophic properties.
  • the GLP- 1(7-36) may subsequently be converted to GLP- 1(7-37) or GLP-l(7-36)amide.
  • GLP-l(7-36) may suitably be converted to GLP-l(7-37) by C-terminal addition of Gly.
  • Such conversion may, for instance, be effected by cleaving the C-terminal Arg36 from GLP-l(7-36) by means of a suitable enzyme, e.g. carboxypeptidase B or carboxypeptidase Y, in the presence of Arg-Gly dipeptide, resulting in replacement of the C-terminal Arg36 by Arg-Gly.
  • GLP-l(7-37) may be produced by processing of the GLP-1 precursor with a suitable enzyme capable of cleaving amino acid sequences at arginine residues (such as one of the enzymes indicated above) in the presence of Gly.
  • GLP-l(7-37) may be prepared by constructing a DNA sequence encoding a GLP-1 precursor containing two or more DNA sequences encoding GLP- 1(7-37) interspersed by sequence(s) coding for at least one arginine residue.
  • the GLP-1 precursor may be prepared by the method of the invention as described above, and in step (e) of the method, the precursor may be processed with a suitable arginine-specific protease such as the mouse submaxillary gland protease and subsequently with carboxypeptidase B (an exopeptidase which cleaves off C-terminal arginine residues one at a time) so as to end processing at the C-terminal glycine.
  • GLP-l(7-36) may suitably be converted to GLP-l(7-36)amide by cleaving Arg36 from GLP-l(7-36) by means of a suitable enzyme, e.g. carboxypeptidase B or carboxypeptidase Y, in the presence of Arg-amide, resulting in the replacement of the C-terminal Arg36 by Arg-amide.
  • the conversion may alternatively be carried out by cleaving the GLP-1 precursor with an Arg-specific enzyme in the presence of NH 3 in a medium which has a reduced water content, e.g. in an organic solvent.
  • GLP-l(7-36) may be derivatives which are chemically modified at the C-terminal carboxylic acid group, e.g. pharmaceutically acceptable lower alkyl esters formed with the C-terminal carboxylic acid group, alkyl meaning e.g. methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl or alkylamide or dialkylamide wherein alkyl is as mentioned above.
  • pharmaceutically acceptable lower alkyl esters formed with the C-terminal carboxylic acid group alkyl meaning e.g. methyl, ethyl, propyl, isopropyl, butyl, or tert-butyl or alkylamide or dialkylamide wherein alkyl is as mentioned above.
  • a cassette of four GLP- 1(7-36) coding units was assembled from synthetic oligonucleotides by polymerase chain reaction (PCR) overlap extension.
  • PCR polymerase chain reaction
  • two separate PCR reactions single stranded oligonucleotide templates were joined.
  • the extended products of these reactions were combined and joined in a third PCR reaction.
  • PCR was performed as described in K.B. Mullis et al. Meth. Enzymol. 155 R. Wu (Ed.), 1987, pp. 335-350 using 10 ⁇ mol oligonucleotide as template, sequence specific oligonucleotide primers and AmpliTaq(TM) DNA polymerase (Perkin Elmer- Cetus).
  • PCR was carried out for 25 cycles of amplification for 1 minute at 94°C, 1 minute at 55°C and 2 minutes at 72°C followed by 10 minutes at 72°C.
  • Oligonucleotides were synthesized on an ABI 394 DNA Synthesizer using phosphoamidite chemistry on a controlled pore glass support (Beaucage and Caruthers (1981) Tetrahedron Letters 22 pp.1859-1869).
  • the sequence of the oligonucleotide primers was the following:
  • the amplified PCR product was digested with restriction endonucleases BamHI and Xbal ( New England Biolabs Inc., Berverly, Ma, USA) and subcloned into the cloning vector pSKII+ ( available from Stratagene Cloning Systems, La Jolla, CA, USA) by the method described in (Sambrook et al. , Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor, 1989).
  • the nucleotide sequence of the PCR product was verified by the dideoxy chain termination metod of F. Sanger et al. PNAS, 74, 1977, pp. 5463-5467, using double stranded plasmid DNA as template and sequence specific oligonucleotides as sequencing primers.
  • the resulting double stranded PCR fragment had the following DNA sequence:
  • oligonucleotide linker harboring sites for restriction endonucleases BamHI, Xhol and Spel was introduced into the BamHI site of expression vector pET3a (available from AMS Biotechnology, UK, Ltd.). Oligonucleotides were synthesized on an ABI 394 DNA Synthesizer using phosphoamidite chemistry on a controlled pore glass support (Beaucage and Caruthers (1981) Tetrahedron Letters 22 pp.1859- 1869).
  • the oligonucleotide linker had the following sequence:
  • the GLP1 cassette was subcloned as a BamHI-Xbal fragment into the BamHI-Spel site of the plasmid construct above by the method described in (Sambrook et al. , Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor, 1989).
  • the resulting expression cassette thus consists of four GLP1 (7-36) coding units preceded by sequences derived from bacteriophage T7 Gene 10 encoding a 15 residue leader peptide.
  • the expression cassette translates into the following peptide sequence:
  • the expression plasmid was introduced into E. coli BL21(DE3)/pLysS (available from AMS Biotechnology, UK, Ltd.) which was made competent according to procedures described in Sambrook et al., op.cit.
  • IPTG Isopropyl (beta)-D-Thiogalactopyranoside
  • the eluting material was collected and the N-terminal amino acid sequence was found by Edman degradation on a Applied Biosystems 470A automatic sequencer, demonstrating that the collected material is pure and has the N- terminal of the GLP-1 precursor.
  • the sequencer was stopped after 21 cycles and the obtained sequence covers 21 amino acids including the prepeptide and part of the first GLP-1 monomer.
  • the result was confirmed by electrospray mass spectrometry analysis (SCIEX API III), that in addition demonstrate that the precursor consists of 4 units of GLP-l(7-36).
  • the collected precursor was found have a molecular mass of 14541 Daltons. The theoretical calculated molecular weight of the entire precursor is 14677.
  • the mass difference is due to a missing methionine residue at the N-terminal of the prepeptide as seen from the amino acid sequence analysis.
  • the methionine residue has most probably been cleaved off by an enzymatic activity from the E.coli. However, the methionine residue is part of the peptide preceding the GLP-1 tetramer and will be cleaved off in the following conversion of the precursor to GLP-1 monomers.
  • a cassette of four GLP-l(7-36) coding units preceded by a methionine codon was made by performing PCR as described (K.B.Mullis op. cit.) using 100 ng of GLP1 teramer harboring plasmid, described in example 1 as 1 template, sequence specific oligonucleotides as primers and AmpliTaq(TM) DNA polymerase (Perkin Elmer- Cetus). PCR was carried out for 25 cycles of amplification for 1 minute at 94°C, 1 minute at 55°C and 2 minutes at 72°C followed by 10 minutes at 72°C.
  • LVKGRHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRHAEGTFTSDVSSYLEG QAAKEFIAWLVKGR The sequence specific oligonucleotides used as primers were the following:
  • Ends of the amplified PCR product were polished using DNA polymerase I large (Klenow) fragment (available from New England Biolabs Inc., Berverly, Ma, USA) as described (Sambrook et al. op. cit.).
  • the PCR product was digested with restriction endonuclease Clal and subcloned into a vector produced by digestion of pHD117-4SP13 (constructed substantially as described in EP 218 651) with restriction endonucleases Clal and PvuII (available from New England Biolabs Inc., Berverly, Ma, USA).
  • the nucleotide sequence of the PCR product was verified by the dideoxy chain termination metod (F. Sanger et al. op.cit.) using double stranded plasmid DNA as template and sequence specific oligonucleotides as sequencing primers.
  • the sequencing primers was the following:
  • the expression plasmid was introduced into E.coli MC1061 (T.V.Huynk et al., in DNA Cloning, vol. 1 (D.M.Glover, Ed.), IRL Press Ltd., Oxford, England, 1983 pp. 56-110) which was made competent according to procedures described in Sambrook et al. op.cit.
  • Lysed cells were sonicated for 3 pulses for 30 seconds at medium output setting on a MSE soniprep 150 sonicator.
  • the sonicate was centrifuged at 12.000 g for 15 min at 4°C.
  • the pellet (inclusion bodies) was washed once in 1/20 culture volumen. Growth medium, lysate and inclusion bodies were kept for further analysis.
  • the samples were injected onto a Vydac 214TP54 column.
  • the column was equilibrated with 30% (v/v) of MeCN in 0.1% (v/v) TFA. After the injection, the' column was eluted with 30% (v/v) MeCN in 0.1% (v/v) TFA for 5 min, whereafter the concentration of MeCN was increased to 50% over 40 min.
  • the flow was 1 ml/min, the temperature was 30°C and the absorbance at 214 nm was recorded.
  • the identity of the GLP-l(7-36) peptide was confirmed by mass-spectrometry analysis as described in example 2.
  • the GLP-1 peptide was isolated from the digest-mixture (30 min) and submitted to mass spectrometry analysis.
  • the molecular weight determined was: 3299 ⁇ 1 compared to the theoretical molecular weight of 3298.7.
  • GLP-1 precursor was dissolved in 2 ml of.35 mM NaOH at 4°C. Glycine was added to the solution to a resulting concentration of 35 mM and the pH was adjusted to 10.1.
  • the trypsin-like protease from Fusarium oxysporum was obtained from Novo Nordisk A/S (Product: SP387) in an aqueous solution containing 20 mg/ml.
  • the digestion was carried out at 37°C by the addition of 1.2 ⁇ l protease solution to the dissolved precursor.
  • the mixture was analyzed by HPLC as described in example 4.
  • the maximum conversion yield obtained after 5 min of digestion was 5.5%. This rather low yield was due to unspecific cleavages of the precursor at other residue than arginine, and can probably be further optimized by the use of a highly purified enzyme preparation.
  • GLP-1 precursor was dissolved in 2 ml 35 mM NaOH at 4° To 0.4 ml of this solution was added 0.15 ml of 35 mM glycine and the pH was adjusted to 11.5.
  • the trypsin used was obtained from Novo Nordisk A/S (Porcine trypsin, batch P9284-30-4, 5200 USP/mg).
  • the enzyme was dissolved in water in a concentration of 2 mg/ml.
  • the GLP-precursor was dissolved in a concentration of 5 mg/ml as described in example 5. The pH was adjusted to 9.0. Endopeptidase Arg-C (Boehringer, cat. no. 269590, lot no. 13170023-16) was dissolved in water to a concentration of 1.0 mg/ml.
  • the digestion was carried out at 37°C by addition of 15 ⁇ l enzyme solution to 300 ⁇ l of pressure solution.
  • the digest mixture was analyzed by HPLC and described in example 4.

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Abstract

Procédé de production d'un peptide I du type au glucagon GLP-1 (7-36) ou d'un de ses analogues dans une bactérie. Ledit procédé comprend: (a) l'insertion, dans un vecteur d'expression approprié, d'une cassette d'expression comprenant un produit de recombinaison d'ADN codant un précurseur de GLP-1 contenant au moins deux séquences d'ADN consécutives codant pour GLP-1 (7-36) ou un de ses analogues, ledit produit de recombinaison d'ADN codant le précurseur de GLP-1 étant précédé par une séquence promoteur régulant l'expression du précurseur de GLP-1, (b) la transformation d'une bactérie appropriée par le vecteur d'expression préparé à l'étape (a), (c) la culture de la bactérie transformée dans des conditions appropriées permettant l'expression du produit de recombinaison d'ADN codant le précurseur de GLP-1, (d) la récupération du précurseur de GLP-1 obtenu de la culture bactérienne, et (e) le traitement du précurseur de GLP-1, afin d'obtenir GLP-1 (7-36) ou un de ses analogues.
PCT/DK1994/000487 1993-12-23 1994-12-22 Procede de production d'un peptide i du type glucagon WO1995017510A1 (fr)

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Cited By (19)

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US5958909A (en) * 1986-05-05 1999-09-28 The General Hospital Corporation Insulinotropic hormones and uses thereof
US6284727B1 (en) 1993-04-07 2001-09-04 Scios, Inc. Prolonged delivery of peptides
US6461834B1 (en) 1998-11-06 2002-10-08 Bionebraska, Inc. Clostripain catalyzed amidation of peptides
WO2003016349A1 (fr) * 2001-07-19 2003-02-27 Shanghai Hua-Yi Bio-Tech Lab Methode de production d'un peptide 1 de type glucagon (glp-1) 7-36 et d'un analogue de glp-1
US6849708B1 (en) 1986-05-05 2005-02-01 The General Hospital Corporation Insulinotropic hormone and uses thereof
EP1513945A2 (fr) * 2002-05-24 2005-03-16 Restoragen, Inc. Methode de production enzymatique universelle de peptides bioactifs
EP1551435A2 (fr) * 2002-05-24 2005-07-13 Restoragen Inc. Methode de production enzymatique de peptides amides glp-1 (7-36)
EP1572720A2 (fr) * 2002-05-24 2005-09-14 NPS Allelix Corp. Procede de production enzymatique de peptides glp-2 (1-34) et glp-2 (1-33)
US7138486B2 (en) 1986-05-05 2006-11-21 The General Hospital Corporation Insulinotropic hormone derivatives and uses thereof
WO2007000118A1 (fr) 2005-06-29 2007-01-04 Changzhou Pharmaceutical Factory Co., Ltd. Fragments polypeptidiques d'exendine 4 et utilisation correspondante
US7329646B2 (en) * 2001-05-10 2008-02-12 Shanghai Huayi Bio-Lab Co., Ltd. Derivatives of the insulinotropic peptide exendin-4 and methods of production thereof
US7569384B2 (en) * 2004-02-09 2009-08-04 Human Genome Sciences, Inc. Albumin fusion proteins
US7829307B2 (en) 2003-11-21 2010-11-09 Nps Pharmaceuticals, Inc. Production of glucagon-like peptide 2
US7847079B2 (en) 2001-12-21 2010-12-07 Human Genome Sciences, Inc. Albumin fusion proteins
US8765915B2 (en) 2006-02-06 2014-07-01 Csl Behring Gmbh Modified coagulation factor VIIa with extended half-life
WO2016210384A2 (fr) 2015-06-25 2016-12-29 Synlogic, Inc. Bactéries manipulées pour traiter des maladies métaboliques
WO2017139708A1 (fr) 2016-02-10 2017-08-17 Synlogic, Inc. Bactéries génétiquement modifiées pour traiter la stéatohépatite non alcoolique (shna)
WO2018172921A1 (fr) 2017-03-20 2018-09-27 Lupin Limited Expression et production à grande échelle de peptides
WO2023045996A1 (fr) * 2021-09-26 2023-03-30 康霖生物科技（杭州）有限公司 Construction d'acide nucléique pour la thérapie génique de maladies associées au métabolisme des hydrates de carbone

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KR100959549B1 (ko) 2001-07-19 2010-05-27 상하이 후아이 바이오테크 랩 글루카곤 유사 펩타이드 1 glp-1(7-36)과 glp-1 유사체를 생산하는 방법
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US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
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EP1551435A4 (fr) * 2002-05-24 2007-08-08 Restoragen Inc Methode de production enzymatique de peptides amides glp-1 (7-36)
EP1572720A2 (fr) * 2002-05-24 2005-09-14 NPS Allelix Corp. Procede de production enzymatique de peptides glp-2 (1-34) et glp-2 (1-33)
EP1513945A4 (fr) * 2002-05-24 2008-12-24 Restoragen Inc Methode de production enzymatique universelle de peptides bioactifs
US8148508B2 (en) 2002-05-24 2012-04-03 Nps Pharmaceuticals, Inc. Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides
EP1513945A2 (fr) * 2002-05-24 2005-03-16 Restoragen, Inc. Methode de production enzymatique universelle de peptides bioactifs
EP1572720A4 (fr) * 2002-05-24 2008-12-24 Nps Allelix Corp Procede de production enzymatique de peptides glp-2 (1-34) et glp-2 (1-33)
US7829307B2 (en) 2003-11-21 2010-11-09 Nps Pharmaceuticals, Inc. Production of glucagon-like peptide 2
US7569384B2 (en) * 2004-02-09 2009-08-04 Human Genome Sciences, Inc. Albumin fusion proteins
US8143026B2 (en) 2004-02-09 2012-03-27 Human Genome Sciences, Inc. Albumin fusion proteins
WO2007000118A1 (fr) 2005-06-29 2007-01-04 Changzhou Pharmaceutical Factory Co., Ltd. Fragments polypeptidiques d'exendine 4 et utilisation correspondante
US8765915B2 (en) 2006-02-06 2014-07-01 Csl Behring Gmbh Modified coagulation factor VIIa with extended half-life
WO2016210384A2 (fr) 2015-06-25 2016-12-29 Synlogic, Inc. Bactéries manipulées pour traiter des maladies métaboliques
WO2017139708A1 (fr) 2016-02-10 2017-08-17 Synlogic, Inc. Bactéries génétiquement modifiées pour traiter la stéatohépatite non alcoolique (shna)
WO2018172921A1 (fr) 2017-03-20 2018-09-27 Lupin Limited Expression et production à grande échelle de peptides
WO2023045996A1 (fr) * 2021-09-26 2023-03-30 康霖生物科技（杭州）有限公司 Construction d'acide nucléique pour la thérapie génique de maladies associées au métabolisme des hydrates de carbone

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