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Host Cells Used For Production of Recombinant Protein

a technology of host cells and recombinant proteins, which is applied in the direction of genetically modified cells, peptide/protein ingredients, peptide sources, etc., can solve the problems of enormous time required for obtaining the gene product (protein), difficult to produce functional proteins from microorganisms, and low amount of production

Inactive Publication Date: 2010-08-12
TAISHO PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019]Preferably, a foreign gene is introduced into the established cell line of the present invention as mentioned above to produce transformed cells, the obtained transformed cells is cultured so as to allow the cells to produce a recombinant protein encoded by the foreign gene, and the produced recombinant protein can be recovered.
[0065]The established cell line of the present invention is able to proliferate in a serum free medium, is able to synthesize and secrete a large amount of protein of interest, and is able to produce a glycoprotein having a sugar chain structure. Accordingly, the established cell line of the present invention is useful as a host cell used for production of a recombinant protein. In particular, using the established cell line of the present invention as a host cell, it becomes possible to produce a large amount of protein of interest at a low cost in the form of the protein having biological activity.

Problems solved by technology

Thus, there may be cases where it is difficult to produce functional proteins from microorganisms.
Thus, even if a gene can be obtained, an enormous time is required for obtaining the gene product (protein).
This causes a bottleneck in many cases.
On the other hand, when a protein of interest is produced using insect cells or cultured animal cells, this is problematic in terms of (1) a low amount of production, (2) a limited range of genes to be expressed, (3) high costs, and the like.
Accordingly, time and effort are required for obtaining a protein to be subjected to screening.
Furthermore, when a large-scale production of proteins is carried out using animal cells, this method has been problematic in the following respects.
However, the amount of a protein that can be produced by a single transfection is limited, and a scale-up is not easy.
Thus, large quantities of cells and transfection reagents are required.
However, this method has been problematic in that it takes a long time (several months) to produce a cell line into which a foreign gene has been stably introduced.

Method used

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  • Host Cells Used For Production of Recombinant Protein
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  • Host Cells Used For Production of Recombinant Protein

Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation of Cat Renal Tubular Epithelial Cells

[0045]Cat distal tubule-derived epithelial cells were isolated as follows according to the method described in Soshiki Baiyo no Gijyutsu (2nd edit) Nihon Soshiki Baiyo Gakkai Hen, Asakura Shoten, 7-4 Jinsaibo Baiyo Ho (pp. 141-145) (issue Culture Techniques (2nd edit) edited by the Japanese Tissue Culture Association, Asakura Shoten, 7-4 Renal Cell Culture Method (pp. 141-145)). A cat was subjected to anesthesia, and the kidney was aseptically excised. The excised kidney was fragmented with a knife in a Joklik buffer (111 mM NaCl, 24 mM NaHCO3, 10 mM Na2HPO4, 5.4 mM KCl, 1 mM Mg2SO4, and 11 mM glucose). 30 ml of a 0.02% collagenase digestive fluid was added to 1 g of the fragmented tissue section in a 100-ml Erlenmeyer flask, and the obtained mixture was then stirred with a stirrer for 30 minutes. Thereafter, a supernatant was discarded, and a fresh digestive fluid was added, followed by a further reaction for 30 minutes. The fragmented...

example 2

Establishment of FKD Cells

[0046]Using Lipofectamine-Plus (Invitrogen), pSV3-neo (ATCC37150) was transfected to the distal tubule-derived cells isolated in Example 1. Forty-eight hours after the transfection, the medium was exchanged with a G418-containing medium, and a stable mutation introduced cell line was then selected. The obtained FKD cells are shown in FIG. 1. The proliferated cells were subjected to a passage, and the resultant cells were then preserved in liquid nitrogen.

example 3

Subculture (Passage) of FKD Cells

[0047]FKD cells were subcultured in a DMEM-F12 (+supplement) medium on a collagen-coated dish at 37° C. in the presence of 5% CO2. When the cells became 80% to 90% confluent, the medium was removed. The cells were washed with PBS / EDTA, and 1 ml of PBS / EDTA was then added thereto, followed by incubation at 37° C. in the presence of 5% CO2 for 5 minutes. Subsequently, the cells were washed with 0.1% trypsin-PBS / EDTA, followed by incubation under the same above conditions. Thereafter, the resultant cells were suspended in a DMEM-F12 (+supplement) medium, and the suspension was diluted and then cultured in a fresh collagen-coated dish. As a supplement (GIBCO ITS-X supplement), a product comprising insulin, transferrin and serine was used.

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Abstract

It is an object of the present invention to provide host cells, which are able to proliferate in a serum free medium, which are able to synthesize and secrete a large amount of protein of interest, and which are able to produce a glycoprotein having a sugar chain structure. The present invention provides an established cell line, which is established from distal tubular epithelial cells derived from a mammal.

Description

TECHNICAL FIELD[0001]The present invention relates to host cells used for production of a recombinant protein, and a use of the aforementioned host cells. The present invention particularly relates to host cells used for production of a recombinant protein established from a mammal, and a use of the aforementioned host cells.BACKGROUND ART[0002]The functional analysis of a gene whose functions are unknown, it is essential to obtain a recombinant protein encoded by the aforementioned gene. The currently used expression systems for production of recombinant proteins include a prokaryotic system, a eukaryotic system, and a cell-free system. Such prokaryotic systems used as hosts include: Escherichia coli and related bacteria (e.g. E. coli, bacteria of genus Pseudomonas, bacteria of genus Zymomonas, bacteria of genus Thermus, etc.); and Bacillus subtilis and related bacteria) (e.g. Actinomycetes, lactic acid bacteria, amino acid-producing bacteria, acetic acid bacteria, thermophilic bac...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N5/06C07K2/00C12N5/071
CPCC12N5/0686C12N2510/02C12N2500/99C12N2500/92
Inventor TAIRA, HIDEHARUYAMASHITA, TETSUROMIYAZAKI, MASAO
Owner TAISHO PHARMACEUTICAL CO LTD
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