Method for the production of polypeptides

A technology of polypeptide chains and heterologous polypeptides, applied in the direction of microorganism-based methods, biochemical equipment and methods, fusion polypeptides, etc., can solve problems such as proteolytic degradation sensitivity, unstable structure, and heterogeneous products

Inactive Publication Date: 2004-05-19
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Another problem encountered with expressing heterologous proteins in yeast is low yields, which may be due to proteolytic processing within intracellular compartments and at the plasma membrane due to aberrant processing at internal sites of the protein, e.g., human thyroid The secretion of pararenaline (Gabrielsen et al., Gene, 90:255-262, 1990; Rokkones et al., J. Biotechnology, 33:293-306, 1994), and the secretion of beta-endorphin in Saccharomyces cerevisiae (Bitter et al. , Proceedings of the National Academy of Sciences of USA, 81:5330-5334, 1984)
Some polypeptides, such as those with chains of about 10-55 amino acids or less, with no or some disulfide bonds, and / or those rich in basic amino acids, such as β-endorphin, glucagon Glucagon and glucagon-like peptides, when expressed in heterologous hosts, may be particularly sensitive to intracellular and extracellular proteolytic degradation due to their short-chain open and unstable structures, resulting in heterogeneous product

Method used

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  • Method for the production of polypeptides
  • Method for the production of polypeptides
  • Method for the production of polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Disruption of the Δyap3::URA3 gene

[0057] The Δura3 deletion mutation was constructed as follows:

[0058] pJJ244 (pUC18 including the 1.2 kb Hind III fragment of the URA3 gene) was digested with StyI, filled in with Klenow polymerase, and self-ligated. The resulting plasmid, called pS194, contains the URA gene with the 84 bp Sty I-Sty I fragment deleted, see figure 1 .

[0059] Δyap3::URA3 gene disruption plasmid pME1389 was constructed as follows:

[0060] The 2.6 kb Sac I-Pst I fragment containing the YAP3 gene in pME768 (Egel-Mitani et al., Yeast 6: 127-137, 1990) was inserted into the 2-6 kb Sac I fragment of pIC19R (Marsh et al., Gene 32: 481-485, 1984). In the l-Pst I fragment, the resulting plasmid was pME834. pME834 was digested with Hind III to delete the 0.7 kb YAP3 fragment and replace it with the 1.2 kb Hind III fragment inserted into the URA3 gene (Rose et al., Gene, 29:113-124, 1984), resulting in the plasmid pME1389. The construction of...

Embodiment 2

[0063] Embodiment 2: Construction of diploid Δyap3 / Δyap3 strain

[0064] ME1487 was mutagenized with EMS (ethyl methanesulfonate), and ura3 mutants were selected on plates containing 5-FOA. and then by using Image 6 One of the selected isolates, ME1656, was transiently transformed with pME973 as shown to undergo a mating type switch (Herskowitz and Jensen, Methods in Enzymology, 194:132-146, 1991). pME973 contains the genes encoding the HO (homophylic complex) endonuclease and URA3 inserted into the YEp13 plasmid (Rose and Broach, Methods in Enzymology, 185:234-279, 1990). Haploid strain ME1695, which had switched from MATα to MATa and had the following genetic background: MATa Δyap3::ura3 pep4-3Δtpi::LEU2 leu2Δura3, was selected from transient transformants.

[0065] ME1695 was then hybridized to ME1487 by micromanipulation (Sherman and Hicks, Methods in Enzymology, 194: 21-37, 1991) to obtain Δyap3 / Δyap3 diploids. From the resulting diploids, line ME1719 was selected wit...

Embodiment 3

[0067] Construction of Δyap3::URA3::Δylr121c Double Disruption Strain

[0068] To construct a one-step gene disruption strain of two tightly linked genes encoding YAP3 and YLR121C, the following two oligonucleotide primers were synthesized:

[0069] P1 length 57bp: YLR121C / URA 3 primer

[0070] 5′-GAT CGA ACG GCC ATG AAA AAT TTG TAC TAG CTA ACG

[0071] AGC AAA GCT TTT CAA TTC AAT-3′

[0072] P2 length 57bp: YAP3 / URA3 primer

[0073] 5′-CCA GAA TTT TTC AAT ACA ATG GGG AAG TTG TCG TAT

[0074] TTA TAA GCT TTT TCT TTC CAA-3′

[0075] P1 and P2 each contain 40 nucleotides corresponding to the sequences in the YLR121C and YAP3 coding regions, and also contain a Hind III restriction site (AAGCTT) and 12 nucleotides corresponding to the flanking sequence of the URA3 gene (YEL021W) acid. Using URA3 gene as a template, PCR was carried out using P1 and P2, and the generated 1248bp PCR fragment contained a URA3 selectable marker, which was flanked by 40 nucleotides derived...

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Abstract

The present invention relates to a novel method for the production of short chain polypeptides, including polypeptides having up to 3 disulfide bonds and / or structures rich in basic amino acid residues, and open structured short chain polypeptides, e.g. glucagon, glucagon like peptides and their functional analogues, in genetically modified yeast cells having reduced activity of YAP3 protease. The invention further comprises genetically modified yeast cells, and a method for the preparation of said yeast cells.

Description

field of invention [0001] The present invention relates to a novel method of producing short-chain polypeptides in genetically modified yeast cells, these polypeptide chains include polypeptides having up to three disulfide bonds and / or having a structure rich in basic amino acid residues, and having an open Structural short-chain polypeptides, such as glucagon, glucagon-like peptide and their functional analogs, said genetically modified yeast cells, and methods for preparing said yeast cells. Background of the invention [0002] For many kinds of polypeptides, such as glucagon, glucagon-like peptides and their functional analogs, after transforming yeast cells with a suitable expression vector containing the DNA sequence encoding the protein, it has been successfully produced in yeast. expression of these heterologous proteins. Because of its stable yield and safety, yeast, especially Saccharomyces cerevisiae, is preferred as a host microorganism for p...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K14/475C07K14/575C07K14/605C12N1/19C12N15/81C12P21/02C12R1/865
CPCC07K14/57509C07K2319/02C07K2319/00C07K14/605C12N15/81C07K14/475
Inventor M·艾格尔-米塔尼J·布兰德特K·瓦德
Owner NOVO NORDISK AS
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