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Double aqueous phase solid-liquid separation method for yeast origin recombinant protein fermentation liquor

A recombinant protein and solid-liquid separation technology, which is applied in the field of bioengineering, can solve the problems of high cost of solid-liquid separation and difficult removal of bacteria, and achieve the effect of simple and easy recovery method, good bacteria removal rate and good separation effect

Inactive Publication Date: 2009-07-15
DALIAN UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of difficult removal of yeast-derived recombinant protein fermentation broth and high cost of solid-liquid separation, and provide a new method for solid-liquid separation of yeast-derived recombinant protein fermentation broth

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The recombinant human serum albumin fermentation broth was obtained by applying the genetically engineered bacteria and production method constructed by our own patent application technology (CN1854301A and CN1854306A), the bacterial concentration was 37g / 100ml fermentation broth, and the protein concentration was 9.5g / L.

[0014] At 4°C, take 200ml of the above-mentioned recombinant human serum albumin fermentation broth, add 63.5g of dipotassium hydrogen phosphate, after the salt is dissolved, add 68.8ml of absolute ethanol, stir well, pH 11, stand at 4°C for 3 hours, extract Phase 160ml, raffinate phase 120ml, the extract phase is clear and transparent, cell removal rate is 99.95%, using Bradford method to measure the protein concentration of the extract phase is 7.6mg / ml, and the protein concentration of the raffinate phase is 0.2mg / ml. The protein recovery rate was 98.0%, and the partition coefficient reached 50.6. SDS-PAGE electrophoresis analysis showed that reco...

Embodiment 2

[0017] Use 200ml of the fermented broth in Example 1, add 40g of sodium carbonate and 65ml of ethanol in sequence at 25°C, mix, and the pH is 13. After stirring and mixing, the two-phase separation operation was carried out according to the operation of Example 1, the protein recovery rate was 86.2%, and the cell removal rate was 99.96%.

Embodiment 3

[0019] Using 200ml of the fermented broth in Example 1, add 15g of sodium chloride, 42g of potassium phosphate, and 68.5ml of ethanol in order at 4°C, stir well, and the pH is 13. After stirring and mixing, the two-phase separation operation was carried out according to the operation of Example 1, the protein recovery rate was 85.2%, and the cell removal rate was 99.96%.

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Abstract

The invention belongs to the technical field of bioengineering and relates to a solid-liquid separation method of yeast-derived recombinant protein fermentation liquor. The solid-liquid separation method is characterized in that the yeast-derived recombinant protein fermentation liquor is added with soluble inorganic salt and a hydrophilic organic solvent and fully stirred, the pH value of a system is regulated by regulating the proportion of the salt, the hydrophilic organic solvent and the fermentation liquor, the operation temperature is controlled to form aqueous a two-phase, an upper phase is an extract phase rich in the recombinant protein, a lower phase is a raffinate phase containing thallus; and the recombinant protein, the organic solvent in the extract phase and the inorganic salt in the raffinate phase are respectively separated and recovered. The solid-liquid separation method solves problems of hard thallus removal, high cost and the like in the existing technology for producing the recombinant protein by a fermentation method, is characterized by good separation effect, short separation time, low cost and being easily enlarged by industrialization, and is the solid-liquid separation method of the yeast-derived recombinant protein fermentation liquor with industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to the solid-liquid separation technology of microbial fermentation liquid, in particular to the solid-liquid separation technology of yeast-derived recombinant protein fermentation liquid. Background technique [0002] Yeast cells, as the production factory of recombinant proteins, have been widely used in the production of recombinant proteins, especially those with complex structures and large quantities, such as recombinant human serum albumin (Microbiology Bulletin, 2003, 30(5): 128-132) and its fusion protein (patent CN200610049070.1), transferrin (progress in biochemistry and biophysics, 2005, 32(7): 625-629), lactoferrin (Chinese dairy industry, 2005, 33(5): 4 -7), industrial enzymes, etc. The solid-liquid separation technology has become one of the bottlenecks in the industrial production of recombinant proteins with yeast. The solid-liquid separation technology of y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 修志龙董悦生贾茜江波田明玉杜力王志明黄顺军高健王冀民李梅彦
Owner DALIAN UNIV OF TECH
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