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Production method for Pichia pastoris expression recombinant human interleukin 11

A technology of Pichia pastoris and human interleukin, applied in the field of large-scale purification of rhIL-11, can solve the problems of unfavorable purification, high production cost, low expression level, etc. Simple to use effects

Active Publication Date: 2013-07-10
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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  • 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 engineering bacterium Pichia pastoris JYIL4 (CCTCC NO: M 204072) is fermented and produced (refer to the published patent, application number 99118279.0), and the present invention adopts a two-step method of fermentation. 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% to indu...

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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 a Chinese patent application filed on November 25, 2004, with the application number 200410089088.5, and the title of the invention is "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 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 [0003] 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/54C07K1/20C07K1/16
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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