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Method of producing protein by using hamster IGF-1

An IGF-1, protein technology, applied in the field of preparing desired recombinant protein, can solve the problems of expensive serum, complex desired protein, pollution and the like

Inactive Publication Date: 2008-03-26
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned operations have the following disadvantages: 1) the relevant mammalian-derived serum is expensive; 2) due to quality differences between batches, stable proliferation cannot be obtained; 3) there is a possibility of contamination by viruses or mycoplasma; and 4 ) Complexity of isolating and purifying the desired protein from the culture medium as a cell product

Method used

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  • Method of producing protein by using hamster IGF-1
  • Method of producing protein by using hamster IGF-1
  • Method of producing protein by using hamster IGF-1

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preparation example Construction

[0166] In the production method of cells used in the production of various desired proteins described above, IGF-1 is preferably the polypeptide of the present invention.

[0167] The present invention further provides a method for screening cells for a desired protein comprising the steps of:

[0168] (a) a step of introducing DNA encoding a desired protein into CHO cells,

[0169] (b) a step of selecting a cell expressing a desired protein among the cells prepared in (a),

[0170] (c) a step of introducing DNA encoding IGF-1 into the cell selected in (b),

[0171] (d) A step of selecting cells expressing a desired protein and IGF-1 among the cells prepared in (c).

[0172] In the above screening method, IGF-1 is preferably the polypeptide of the present invention.

[0173] The gene encoding IGF-1 used in the method for preparing cells for producing a desired protein or the method for screening cells for producing a desired protein in the present invention is not particula...

Embodiment 1

[0177] Example 1: Cloning of cDNA encoding hamster IGF-1

[0178] (1) Synthesis of cDNA

[0179] Total RNA was extracted from anti-IL-6R antibody-producing cells in which an anti-interleukin-6 receptor (IL-6R) antibody gene was introduced into CHO-DXB11 cells (JP-A-8-99902, Reference Example 2). cDNA is synthesized from the resulting mRNA.

[0180] Specifically, proceed as follows. After 72 hours after passaging, about 5×10 cells in the proliferating phase 6 The CHO cells were centrifuged at 4°C and 1000rpm for 5 minutes, and the cells in the precipitated state were obtained at the bottom of the centrifuge tube without culture medium. Add 4ml of Sepasol-RNAI preheated at 65°C, and then use a pipette to quickly To dissolve, let stand at room temperature for 5 minutes. Further, 0.8 ml of chloroform was added, stirred well with a Voltex, and then left to stand at room temperature for 3 minutes. By cooling the centrifuge Avanti TM After J25 (manufactured by BECKMAN) was cen...

Embodiment 2

[0186] Example 2: Determination of the base sequence and amino acid sequence of the cDNA encoding hamster IGF-1

[0187] The gene sequence of the cloned gene was determined by a DNA sequencer, and the amino acid sequence was further determined, confirming that it encodes hamster IGF-1. The obtained amino acid sequence is shown in FIG. 1 and SEQ ID NO: 1, and the DNA sequence is shown in SEQ ID NO: 2.

[0188] The mature secreted IGF-1 of each species is composed of 70 amino acids, but the hamster IGF-1 gene has a specific sequence. Compared with human IGF-1, the 20th P (human: D), the 35th P I (human: S), the three amino acids of T (human: A) at the 67th position are different from human acids (Figure 2)

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Abstract

A polypeptide selected from the group consisting of (1) a polypeptide having the amino acid sequence represented by SEQ ID NO:1 and (2) a polypeptide having an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by substitution of the amino acid at the 3-position by another amino acid and having an insulin-like growth factor 1 (IGF-1) activity; a DNA encoding the above polypeptide; a vector containing the above DNA; a host cell containing the above vector; and a method of producing a recombinant protein by using the above polypeptide.

Description

technical field [0001] The present invention relates to novel hamster IGF-1 polypeptides. The present invention further relates to methods for efficiently and inexpensively producing desired recombinant proteins using the above-mentioned novel IGF-1 polypeptides. Background technique [0002] When using genetic recombination technology to produce proteins for pharmaceuticals, animal cells are often used as host cells for the production of recombinant proteins because complex post-translational modifications or folding that cannot be achieved in bacterial cells can be achieved using animal cells. [0003] Currently, when culturing animal cells to obtain natural proteins produced by the animal cells, or culturing animal cells into which a gene encoding a desired protein is introduced to produce a desired protein, etc., in addition to salts, sugars, amino acids, and vitamins, In addition to such basic nutrients, in order to proliferate the animal cells, it is necessary to add ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K16/26C12N15/18C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02C12Q1/02A61K38/27A61P3/06A61P19/08A61P21/00C12N5/07C12N5/071
CPCC07K16/2866C12P21/02C07K14/65A61P19/08A61P21/00A61P3/06C07K14/62C07K16/26C12N15/11A61K38/27
Inventor 田渊久大田中佐依子
Owner CHUGAI PHARMA CO LTD
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