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Novel tools for the production of glycosylated proteins in host cells

A cell and organelle technology, applied in the field of glycoprotein manufacturing and protein glycosylation engineering in eukaryotes, which can solve the problems of low therapeutic value, virus contamination, and complicated strain design.

Inactive Publication Date: 2011-09-28
LONZA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of currently used mammalian expression systems for the production of recombinant proteins are (1) low yields, (2) expensive fermentation procedures, (3) complex strain design, and (4) risk of viral contamination
In particular, therapeutic glycoproteins produced in yeast may elicit unwanted immune responses in higher eukaryotes, especially animals and humans, resulting in low therapeutic value of therapeutic proteins produced in yeast and its ilk

Method used

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  • Novel tools for the production of glycosylated proteins in host cells
  • Novel tools for the production of glycosylated proteins in host cells
  • Novel tools for the production of glycosylated proteins in host cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0868] Embodiment 1 has the manufacture of the glycoprotein of Man3GlcNAc2 structure

[0869] 1.1 Yeast medium and method

[0870] All strains were grown on YPD medium unless otherwise stated. Strain YG1137 was maintained on YPGal. Strains YCN1 (Δrft1 ), YG1363 (Δalg3Δalg11 ), YG1365 (Δalg11 ), and YG1830 (alg2-1 ) were grown in medium supplemented with 1M sorbitol, unless otherwise stated.

[0871] 1.2 Strain Construction

[0872] The entire Alg11 open reading frame was replaced in SS328XSS330 by integrating the PCR product including the S. cerevisiae HIS3 site. Transformed yeast strain YG1141 (MATa / αade2-201 / ade2-201 ura3-52 / ura3-52 his3Δ200 / his3Δ200 tyr1 / +lys2-801 / +Δalg11::HIS3 / +) was sporulated and tetrads were split to Δalg11 haploid was obtained, and YG1361 (MATαade2-201 ura3-52 his3Δ200Δalg11::HIS3) paired with YG248 (MATaΔalg3::HIS3 ade2-101 his3Δ200 lys2-801 ura3-52). The resulting diploid YG1362 (MATa / αade2-201 / ade2-201 ura3-52 / ura3-52 his3Δ200 / his3Δ200 lys2-8...

Embodiment 2

[0924] Example 2 Complex system for glycosylation

[0925] 2.1 Expression of novel LLOs and protozoan oligosaccharyltransferases in yeast mutant strains

[0926] In a preferred embodiment, there is provided a complex system for protein glycosylation, especially in yeast, comprising at least 3 entities: (i) a lipid as a precursor of an oligosaccharyltransferase Links the production of Man3GlcNAc2; (ii) a flippase such as (Flc2'); and (iii) a protozoan oligosaccharyltransferase (POT) that exhibits relaxed substrate specificity.

[0927] In order to combine the two heterologous proteins, flippase and protozoan oligosaccharyltransferase, a vector comprising both parts was constructed.

[0928] As a result, protozoan oligosaccharyltransferase (LmStt3D) under the regulation of GPD promoter and cyc1 terminator was inserted into the vector including Flc2' as a reverse transcribed gene. Plasmids carrying LmStt3D, Flc2' or both enzymes were transformed into wild-type yeast (YG1509) ...

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Abstract

The invention improves glycoprotein production and protein glycosylation engineering in eukaryotes, specifically the production of human-like complex or hybrid glycosylated proteins in lower eukaryotes such as yeasts. The invention provides glycosylation modified eukaryotic host cells capable of producing glycosylation optimized proteins useful as immunoglobulins and other therapeutic proteins, and provides cells capable of producing glycoproteins having glycan structures similar to glycoproteins produced in human cell. The invention further provides proteins with human-like glycan structures and novel compositions thereof producible by these cells.

Description

technical field [0001] The present invention relates to the fields of glycoprotein production and protein glycosylation engineering in eukaryotes, in particular to the production of human-like complexes or hybrid glycosylated proteins in lower eukaryotes such as yeast. The invention further relates to glycosylation-modified eukaryotic host cells capable of producing glycosylation-optimized proteins that are particularly effective in humans as immunoglobulins and other therapeutic proteins. The invention also relates to genetically engineered eukaryotic non-human cells capable of producing glycoproteins having a polysaccharide structure similar to those produced in human cells. Accordingly, the present invention further relates to proteins having a human-like polysaccharide structure and novel compositions thereof which can be produced by said cells. Background technique [0002] Most protein-based biopharmaceuticals expose some form of post-translational modification that c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/395C12P21/00C12N15/81C12N9/10C07K14/44
CPCC12P21/005C12N9/1048C12N9/1081C12Y204/99018
Inventor 乔尼·赫勒纽斯克里斯廷·纽珀特马库斯·艾比法劳西·帕尔赛-纳赛比亚历山大·丹尼尔·弗雷
Owner LONZA LTD
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