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Method for producing recombinant protein

A technology of recombinant protein and recombinant cells, applied in the direction of microorganism-based methods, biochemical equipment and methods, tissue culture, etc., to achieve the effect of improving productivity

Pending Publication Date: 2021-12-17
SPIBER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although improvement was attempted in this way, a system with higher productivity still needs to be established

Method used

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  • Method for producing recombinant protein
  • Method for producing recombinant protein
  • Method for producing recombinant protein

Examples

Experimental program
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preparation example Construction

[0030] The method for producing a recombinant protein according to the present embodiment includes the following steps: a first step, which comprises galactose, and a protein selected from the group consisting of mannitol, raffinose, and culturing in a medium containing at least two of the group consisting of melibiose; the second step is to further culture the recombinant cells under conditions that induce the expression of an inducible promoter after the first step.

[0031] (Recombinant protein)

[0032] Examples of the recombinant protein produced by the production method according to the present embodiment (hereinafter also referred to as "target protein") include any protein preferably produced on an industrial scale, for example, industrially usable proteins, available Medical proteins, structural proteins, etc. Specific examples of proteins that can be used in industry or medicine include spider silk proteins, enzymes, regulatory proteins, receptors, peptide hormones,...

preparation example 1

[0094] (Preparation Example 1: Prepare Modified Silk Fibroin by Chromosomal Integration Expression Strain)

[0095] (1-1) Preparation of modified fibroin expression strain (chromosomal integration expression strain)

[0096] According to the base sequence and amino acid sequence of silk fibroin (GenBank accession number: P46804.1, GI: 1174415) derived from Nephila clavipes, a modified silk heart having the amino acid sequence shown in SEQ ID NO: 1 was designed. protein (hereinafter also referred to as "PRT410").

[0097] The amino acid sequence (Met-PRT410) shown in SEQ ID NO: 2 is a sequence in which amino acid residues were substituted, inserted, and deleted for the amino acid sequence of silk fibroin derived from the spider spider for the purpose of improving productivity. The amino acid sequence represented by SEQ ID NO: 1 (PRT410) is an amino acid sequence obtained by adding the amino acid sequence represented by SEQ ID NO: 3 (tag sequence and hinge sequence) to the N-te...

preparation example 2

[0132] (Preparation example 2: prepare modified silk fibroin by plasmid type expression strain)

[0133] (2-1) Preparation of modified fibroin expression strain (plasmid expression strain)

[0134] According to the base sequence and amino acid sequence of silk fibroin (GenBank accession number: P46804.1, GI: 1174415) derived from Nephila clavipes, a modified silk heart having the amino acid sequence shown in SEQ ID NO: 4 was designed. protein (hereinafter also referred to as "PRT918").

[0135] The amino acid sequence (Met-PRT918) shown in SEQ ID NO: 5 is a sequence in which amino acid residues were substituted, inserted, and deleted for the amino acid sequence of silk fibroin derived from the spider spider for the purpose of improving productivity. The amino acid sequence represented by SEQ ID NO: 4 (PRT966) has the amino acid sequence represented by SEQ ID NO: 3 (tag sequence and hinge sequence) added to the N-terminus of the amino acid sequence represented by SEQ ID NO: 5....

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Abstract

The present invention pertains to a method for producing a recombinant protein, said method comprising: a first step for culturing a recombinant cell, which expresses the recombinant protein under the control of an inducible promoter, in a medium containing galactose and at least two substances selected from the group consisting of mannitol, raffinose and melibiose; and a second step for, after the first step, further culturing the recombinant cell under such conditions that the expression from the inducible promoter is induced.

Description

technical field [0001] The invention relates to a preparation method of recombinant protein. Background technique [0002] In the industrial production of useful substances using microorganisms, improvement of productivity is an important issue. Therefore, various culture methods have been studied. In the production of recombinant protein, the usual method is to introduce the expression cassette of recombinant protein under the control of an inducible promoter into host cells, and introduce the recombinant cells into the culture medium, and after culturing, the cells will be activated by the supply of nutrients. The recombinant cells are proliferated to a desired concentration, and the expression of the recombinant protein is induced by adding an inducer such as arabinose, lactose, or isopropyl-β-thiogalactoside (IPTG) to the promoter. [0003] For example, when a T7 promoter is used for expression of a recombinant protein, an IPTG-induced pET system in combination with T7...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P21/02
CPCC12P21/02C07K14/43518C12R2001/19C12N1/20C12R2001/07C12N2500/34
Inventor 小林雅人仓知建始森英词野田昂文
Owner SPIBER INC
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