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Method for improved high secretory production of proteins

A protein and protease technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, peptides, etc., to achieve high safety effects

Pending Publication Date: 2017-08-18
DAIICHI SANKYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] As mentioned above, although various methods have been suggested for the high-secretion production of proteins in yeast, in general, when transforming host cells by gene introduction, gene disruption, etc., the cells are subject to some kind of stress, so only by introducing several Combinations of existing methods may not be able to achieve synergistic or additive effects
In addition, reports of high secretory production of target proteins by combining the above-mentioned various methods with the above-mentioned various partners are still unknown.

Method used

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  • Method for improved high secretory production of proteins
  • Method for improved high secretory production of proteins
  • Method for improved high secretory production of proteins

Examples

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preparation example Construction

[0130] 2. Protein Preparation

[0131] The protein in the present invention can be produced by culturing the transformed yeast of 1. by a known method, extracting it from the culture, and then purifying it. "Culture" means, in addition to the culture supernatant, any of cultured cells, cultured cells, or disrupted products of cells or cells.

[0132] When the host cell is yeast, as the medium to be cultured, as long as it contains a carbon source, a nitrogen source, inorganic salts, etc. that can nourish the yeast and can effectively perform the culture of yeast transformants, a natural medium can be used , any one of synthetic media. As the carbon source, carbohydrates such as glucose, fructose, sucrose, and starch; organic acids such as acetic acid, lactic acid, citric acid, and propionic acid; and alcohols such as methanol, ethanol, propanol, and glycerin can be used. As a nitrogen source, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, am...

Embodiment 1

[0145] [Example 1] Construction of foreign gene expression vector

[0146] (1) NBRC10746 ( O. minuta , (BiologicalResource Center, NITE)) Construction of a foreign gene introduction vector consisting of aox1 gene promoter and terminator cassette

[0147] AOX1 of NBRC10746 (GENBANK accession number: AB242209) consists of 663 amino acids encoded by a 1992bp nucleotide sequence (base sequence) (SEQ ID NO: 27, SEQ ID NO: 28). Genomic DNA of NBRC10746 prepared by Y-DER Yeast DNAExtraction Reagent (Y-DER Yeast DNA Extraction Reagent) (PIERCE Company, 78870) was used as a template, and primer HdAOXp Fw (5'-GCAAGCTTTCTTTCGCAAACAGCTCTTTG-3':SEQ ID NO: 1) PCR with primer AOXp rv (5'-GAACCCGGGAACAGAATCTAGATTTTTTCGTAAGTCGTAAG-3': SEQ ID NO: 2) [(10 seconds at 98°C, 5 seconds at 55°C, 15 seconds at 72°C) × 30 cycles], using PrimeSTARMax DNA polymerase (Takara Bio, RO45A) was used to amplify the DNA fragment containing the aox1 promoter region including the aox1 promoter region of about ...

Embodiment 2

[0149] [Example 2] Production of ura3 gene disrupted strain

[0150] (1) Preparation of DNA fragments for ura3 gene disruption

[0151] The gene disruption pattern of the DNA fragment using the promoter and terminator of the ura3 ORF is shown in figure 1 . URA3 of NBRC10746 (GENBANK accession number: AB242207) consists of 265 amino acid sequences encoded by a 798bp nucleotide sequence (SEQ ID NO: 29, SEQ ID NO: 30). Genomic DNA of NBRC10746 modulated by Y-DER Yeast DNA Extraction Reagent (PIERCE Company, 78870) was used as a template, primer dURA Fw (5'-GGTACCAGTACTGGAAA-3': SEQ ID NO: 5) and primer dURA rv (5' -CAGATAAACAGGCGACTTTTCGGGTCACGTGACT-3': SEQ ID NO: 6) PCR [(10 seconds at 98°C, 5 seconds at 55°C, 5 seconds at 72°C) × 30 cycles] using PrimeSTAR Max DNA polymerase (Takara Bio, RO45A ), a ura3 terminator region-containing DNA fragment comprising a ura3 terminator region of about 0.5 kbp and a ura3 promoter region of 17 bp was amplified. In addition, PCR using the...

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Abstract

The invention provides a method for improved high secretory production of proteins. The present invention addresses the problem of providing a production system which achieves, in a host cell such as yeast, high secretory production of proteins, particularly proteins having a complex structure such as an S-S bond and which is so safe as to obviate explosion-proof equipment and is suitable for industrial production. The present invention provides a transformed yeast obtained by introducing a chaperone gene and disrupting aox1 and / or protease genes, and a method for producing proteins using the transformed yeast.

Description

technical field [0001] The present invention relates to methods for the high secretion production of proteins in yeast. Background technique [0002] Protein pharmaceuticals such as therapeutic proteins and antibody drugs have rapidly expanded their market due to the development of gene recombination technology, and animal cells such as CHO and NS0; insect cells such as silkworm or SF9 have been used as hosts for the production of these protein pharmaceuticals. ; Escherichia coli, yeast and other microorganisms. Among them, yeast is widely used as a system capable of secreting and producing useful proteins in a relatively inexpensive medium because it can be cultured at a high density. [0003] However, when the amino acid region of the signal sequence present at the N-terminal of the secreted protein is recognized by a signal recognition particle (SRP: signal recognition particle), the secreted protein enters the endoplasmic reticulum through the translocon, and when the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/19C12P21/02
CPCC12P21/02C12N15/09C12N15/62C12N15/81C07K14/395C12N9/0008C12N9/60C12P21/00C12Y102/03001C12Y304/22C12N15/113
Inventor 野中浩一铃木健之津田将志马场悟史市川公久千叶靖典横尾岳彦伊藤理惠
Owner DAIICHI SANKYO CO LTD
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