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Production method for Pichia pastoris expression recombinant human interleukin 11

A technology for Pichia pastoris and human interleukin, which is applied in the field of large-scale purification of rhIL-11, can solve the problems of low expression amount, high production cost, unfavorable purification and the like, achieves simple operation, reduces pollution and loss, and is beneficial to large-scale the effect of industrial production

Active Publication Date: 2012-01-25
HANGZHOU JIUYUAN GENE ENG
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  • Application Information

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Problems solved by technology

However, this system has the following disadvantages: (1) the expression level is not high, (2) the production cost is too high, (3) the protein purification process is complicated
However, this method needs to centrifuge the fermentation broth and concentrate it by ultrafiltration and other methods to remove impurities in the fermentation broth, reduce the conductivity, and then purify the protein by sequential combination of ion exchange chromatography, hydrophobic chromatography and gel chromatography. It is relatively complicated. After ion exchange chromatography, the content of impurity proteins and endotoxins is still relatively high, which is not conducive to subsequent purification

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  • Production method for Pichia pastoris expression recombinant human interleukin 11
  • Production method for Pichia pastoris expression recombinant human interleukin 11
  • Production method for Pichia pastoris expression recombinant human interleukin 11

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Embodiment Construction

[0019] The engineered strain Pichia pastoris JYIL4 (CCTCC NO: M 204072) is fermented (refer to the published patent, application number 99118279.0). The present invention adopts two-step fermentation. In the first stage, the engineered bacteria are inoculated in a semi-synthetic medium with carbon sources such as glycerol or glucose (the formula is: sodium hexametaphosphate 25g / L, CaSO 4 ·2H 2 O 1.176g / L, K 2 SO 4 18.2g / L, MgSO 4 7.28g / L, EDTA 0.925g / L, glycerol 40g / L, PTM1 4.35ml / L, (NH 4 ) 2 SO 4 9g / L, bubble enemy 0.02%. The formula of PTM1 is: CuSO 4 ·5H 2 O 6g / L, NaI 0.08g / L, MnSO 4 ·2H 2 O 3g / L, Na 2 MoO 4 ·2H 2 O 0.2g / L, boric acid 0.02g / L, biotin 0.2g / L, CoCl 2 0.5g / L, ZnCl 2 20g / L, FeSO 4 ·7H 2 O 65g / L, sulfuric acid 5ml) incubate for 16-28 hours until glycerol or glucose is exhausted, and the bacterial density reaches greater than OD 600 =160, this is the growth period of the bacteria. In the second stage, methanol is fed to a concentration of 0.5%-5% to induce ex...

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Abstract

The invention discloses a production method for Pichia pastoris expression recombinant human interleukin 11, which comprises the step of combined purification sequentially through reverse phase column chromatography, hydrophobic column chromatography and gel column chromatography. By adopting the production method, high-purity medicinal recombinant human interleukin 11 proteins can be prepared ina large scale.

Description

[0001] This application is a divisional application of the Chinese patent application filed on November 25, 2004, with the application number 200410089088.5 and the invention title "Method for producing recombinant human interleukin 11 expressed by Pichia pastoris". Technical field [0002] The present invention relates to the application of recombinant DNA technology to produce genetically engineered protein drugs, in particular to a large-scale purification of rhIL-11 from high-density fermentation broth of Pichia pastoris engineering bacteria expressing recombinant human interleukin 11 (rhIL-11) Methods. Background technique [0003] Human Interleukin 11 (hIL-11) is a cytokine that interacts with many hematopoietic cells. It was first identified in the conditioned medium of the mammalian bone marrow cell line PU-34 stimulated by IL-1a. Substance, which can maintain the survival of cultured blood cells for a long time, and then clone hIL-11cDNA from human embryonic fibroblast ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C07K1/20C07K14/54
Inventor 黄岩山马国昌杨志愉李辉孙汉栋徐飞虎周金宝
Owner HANGZHOU JIUYUAN GENE ENG
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