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Method for high efficiency expressing destination protein

A high-efficiency expression and recombinant protein technology, applied in chemical instruments and methods, cells modified by introducing foreign genetic material, peptides, etc. Relative quantity and other issues, to achieve the effect of improving the yield of target protein, good application value and simple method

Inactive Publication Date: 2006-11-29
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Abstract
  • Description
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Problems solved by technology

However, some studies have shown that the production of recombinant proteins cannot be increased to the desired level only by optimizing the culture temperature
(Yoon SK, Song JY, Lee GM. Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells. Biotechnol Bioeng. 2003, 82(3): 289-98) This is because of lowering the culture temperature It can increase the specific production rate of the target protein, but at the same time lead to the inhibition of cell proliferation and reduce the relative number of cells, thus affecting the increase of the target protein production

Method used

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  • Method for high efficiency expressing destination protein
  • Method for high efficiency expressing destination protein
  • Method for high efficiency expressing destination protein

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0018] Use a two-stage culture method to improve the fusion protein (HFI) in recombinant CHO cells

[0019] Expression

[0020] 1. Material:

[0021] A CHO cell line stably expressing the fusion protein HFI (Shi, M., Xie, Z., Feng, J., Sun, Y., Yu, M., Shen, B., Guo, N., 2003. A recombinant anti -ErbB2, scFv-Fc-IL-2 fusion protein retains antigenspecificity and cytokine function. Biotechnol. Lett. 25, 815-819); DMEM medium and protein-free medium HyQSFM4CHO are produced by HyClone; goat anti-human IgG, horseradish Peroxidase-labeled goat anti-human IgG was purchased from Beijing Zhongshan Biotechnology Company.

[0022] 2. Methods and results

[0023] The recombinant CHO cell line stably expressing the fusion protein anti-erbB2-ScFv-Fc-IL-2 was divided into 2×10 6 / Number of bottles, inoculated on the bottom area of ​​25cm 2 There are a total of 16 flasks in the culture flasks. The applied medium is DMEM+10% FBS. After the cells are cultured at 36.5°C-37.8°C t...

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Abstract

This invention discloses method of high efficiency expressing target albumen. Amalgamation albumen expression quantity can be greatly improved by using two steps culture strategy, and the culture temperature of the expression target albumen CHO cell is stabilized. The method in this invention is simple, and easy to realized, its efficiency is high, and cost is not improved. It has good application value not only to laboratory using or commercialization generating.

Description

Technical field [0001] The invention relates to a method for efficiently expressing a target protein. Background technique [0002] The fusion of antibody molecular fragments with other proteins can obtain fusion proteins with multiple biological functions. The genes of enzymes, toxins, cytokines and other biologically active substances can be fused with antibodies to target these biological activities to specific target sites. It can effectively exert biological functions and reduce toxic and side effects. The fusion of antibody fragments with cytokines is an effective means for targeted treatment of tumors, and such fusion proteins are called "immune cytokines." At present, the production of immune cytokines has become the biggest obstacle restricting their large-scale clinical application. In most literature reports, the output of immune cytokines is less than 20μg / ml, which is difficult to meet the requirements of large-scale production. So far, only one immune cytokine has b...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N5/10C12P21/02C07K19/00
Inventor 施明郭宁沈倍奋
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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