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Method for producing Pichia anomala expression recombination human interleukin 11

A production method, the technology of Pichia pastoris, which is applied in the direction of interleukin, animal/human protein, cytokine/lymphokine/interferon, etc., can solve the problems of high production cost, low expression level, unfavorable purification, etc., and achieve Reduce pollution and loss, simple operation, beneficial to large-scale industrial production

Inactive Publication Date: 2005-09-21
HANGZHOU JIUYUAN GENE ENG
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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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  • Method for producing Pichia anomala expression recombination human interleukin 11
  • Method for producing Pichia anomala expression recombination human interleukin 11
  • Method for producing Pichia anomala expression recombination human interleukin 11

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Embodiment Construction

[0018] The engineering bacterium Pichia pastoris JYIL4 (CCTCC NO: M204072) is used for fermentation production (refer to the published patent, application number 99118279.0), and the present invention adopts a two-step fermentation method. In the first stage, the engineered bacteria are inoculated in a semi-synthetic medium (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) cultivated for 16-28 hours until glycerol or glucose was exhausted, and the bacterial density reached greater than OD 600 =160, this is the growth period of the strain. In the second stage, methanol was fed to a concentration of 0.5%-5% t...

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Abstract

The invention discloses a method for producing Pichia anomala expression recombination human interleukin 11 which consists of reversed phase column chromatography, hydrophobic column chromatography, gel column chromatography sequent combination purification. The method can be applied for mass preparation of high purity medicinal recombination human interleukin 11 proteins.

Description

technical field [0001] The present invention relates to the application of recombinant DNA technology to produce genetically engineered protein medicine technology, in particular to a large-scale purification of rhIL-11 from the high-density fermentation liquid of Pichia pastoris engineering bacteria expressing recombinant human interleukin 11 (rhIL-11) Methods. Background technique [0002] Human Interleukin 11 (hIL-11) is a cytokine that interacts with many hematopoietic cells, and was first identified for its activity in the conditioned medium of the IL-1a-stimulated mammalian myeloid cell line PU-34 Substances that can maintain the viability of cultured blood cells for a long time were subsequently cloned into hIL-11 cDNA from a human embryonic fibroblast cell line. Natural hIL-11 mature protein contains 178 amino acids, molecular weight 19Kd; does not contain cysteine, thus lacks disulfide bond, but the protein structure is quite stable; no glycosylation modification; ...

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Application Information

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IPC IPC(8): C07K1/16C07K14/54
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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