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Technique for separating and purifying recombination human serum albumin and fusion protein thereof

A technology of human serum albumin and fusion protein, which is applied in the field of purification of human serum albumin and human serum albumin fusion protein, can solve the problems of not being able to purchase and trial, and not mentioning the purification of human serum albumin fusion protein, etc. Achieve the effect of simple purification steps

Active Publication Date: 2012-08-08
TIANJIN SINOBIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the very important and key weak cationic medium that can withstand high salt is proprietary and cannot be purchased and tried from commercial channels.
At the same time, whether this medium can be used for the purification of human serum albumin fusion protein is not mentioned in the published application

Method used

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  • Technique for separating and purifying recombination human serum albumin and fusion protein thereof
  • Technique for separating and purifying recombination human serum albumin and fusion protein thereof
  • Technique for separating and purifying recombination human serum albumin and fusion protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: High-density fermentation of engineering strains expressing rHSA and its fusion protein Pichia pastoris

[0038]The recombinant human serum albumin engineering expression bacteria used in the present invention, the construction of fusion protein engineering expression bacteria formed by various human serum albumin and therapeutic proteins directly or through linking peptides are all obtained from the Chinese authorized invention patent ZL02, ZL2004100, ZL2007100 and US authorized invention patent US7244 and US invention patent application US2006.

[0039] The rHSA / therapeutic protein fusion protein strain involved therein can utilize the same production process to obtain the expression product. It is generally described as, 1ml of the frozen strain of Pichia pastoris high-expression engineered bacteria is inoculated in 100ml of inorganic salt medium, and cultured at 30°C for 20 hours at 300rpm to reach OD 600 =~11. The average inoculation in five 200ml inor...

Embodiment 2

[0040] Example 2: Vertebrate cells are used to express human serum albumin / EPO fusion protein

[0041] Recombinant cell clones obtained by transfecting adherent CHO cells (CHO-H) or CHO suspension cells (Invitrogen CHO-S cell line) with pCMV-HSA or pCMV-HSA / EPO plasmids, using Neomycin as selection pressure The progeny were cultivated and amplified with serum-free medium (commercially available). In the production stage, 3 days after cell inoculation, continuous culture is started, and serum-free culture supernatant containing human serum albumin or human serum albumin EPO cell culture is collected. At this time, the content of the recombinant protein product in the cell culture medium is above 90mg / L, the pH=7.0, and the conductivity value is between 5-20ms / cm.

Embodiment 3

[0042] Embodiment 3: yeast high-density fermentation post-treatment

[0043] Yeast high-density fermentation broth is pumped into the plate-and-frame filtration system to remove the bacteria to obtain a clarified supernatant. The supernatant can be directly used for separation and purification procedures after suction filtration through a 0.45uM tube filter. The fermentation broth can also be added with active carbon to 0.4-5% after the plate and frame filtration is completed. After mixing well, the activated carbon was removed by a continuous flow high-speed tubular centrifuge to reduce the pigment content in the fermentation broth. After activated carbon treatment, the fermentation supernatant was filtered through a 0.45uM filter before entering the purification procedure. The culture medium containing the target protein obtained from culturing CHO cells in serum-free medium was processed in a continuous-flow high-speed tubular centrifuge, sterilized by a 0.45uM membrane, ...

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Abstract

The invention provides a technological flow with versatility and high efficiency for reforming human serum albumins and fusion proteins of human serum albumins by separating and purifying target proteins. The steps of the method are as follows: the inorganic salt culture medium is used to acquire the genetic engineering microzyme fermented supernatant containing target proteins, and the serum-free culture medium is used to acquire a culture solution of reforming vertebrate cells, and ion exchange type affinity column chromatography medium Capto MMC fillers with higher salinity resistance and high capacity are directly used for separation and purification. The technological flow of the invention has the advantages of simple purification program step, uniqueness, low costs and convenient industrialization.

Description

technical field [0001] The invention belongs to the technical field of protein separation and purification, in particular to the purification of recombinant human serum albumin and human serum albumin fusion protein. The present invention uses specific column chromatography steps, especially the Capto-MMC filler is applied to the purification of recombinant proteins under various salt concentrations (especially high salt concentrations), and is applicable to laboratories, pilot scales and large-scale industrialization Efficient isolation and purification of recombinant proteins related to human serum albumin in production. Especially the large-scale production application of recombinant human serum albumin and human serum albumin fusion protein secreted and expressed by yeast or passaged vertebrate cells. The purification step of the invention is simple, and has uniqueness, universality and unexpected effects. Background technique [0002] Human serum albumin (HSA) is the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/18C07K1/20C07K19/00
Inventor 于在林富岩
Owner TIANJIN SINOBIOTECH LTD
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