Method for purifying recombinant protein

A recombinant protein and purification method technology, which is applied in the preparation methods of peptides, chemical instruments and methods, single-component protein rayon, etc., can solve problems such as gelation, and achieve the effect of fewer steps and high purity

Inactive Publication Date: 2019-06-04
SPIBER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this method, the target protein can be purified to about 70%, but there is a problem that the target protein is limited to hydrophilic recombinant proteins with a hydropathic index (HI) of 0 or less
In addition, depending on the protein, gelation may occur due to the influence of water-based solvents such as water added to remove impurities

Method used

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  • Method for purifying recombinant protein
  • Method for purifying recombinant protein
  • Method for purifying recombinant protein

Examples

Experimental program
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Effect test

preparation example Construction

[0212] (1) Preparation of Polypeptide Solution (Spinning Dope) As described above, the second soluble fraction in which the recombinant protein is dissolved in step (D) does not require further purification steps and can be used as a so-called spinning solution in step (D). (E) Spinning. The viscosity suitable for spinning is usually 10 to 10000 cP (centipoise), and the viscosity can be measured using, for example, a trade name "EMS viscometer" manufactured by Kyoto Denshi Kogyo Co., Ltd. In the case where the viscosity of the second soluble fraction obtained in the step (D) is not in the range of 10 to 10000 cP (centipoise), the viscosity of the second soluble fraction may be adjusted to a viscosity capable of spinning. The viscosity can be adjusted by using the aprotic polar solvents exemplified as preferable solvents in the description of the step (A). The aprotic polar solvent may contain the preferred inorganic salts exemplified in the description of step (A).

[0213] ...

Embodiment 1

[0252] [Example 1 Influence of Inorganic Salt and Temperature in Dissolution Step of Host Cell-derived Protein]

[0253] In the step of dissolving the host cell-derived protein, the addition of the inorganic salt to the first aprotic polar solvent, the concentration of the addition, and the influence of the temperature were examined. Add 1 mL of DMSO containing 0M, 0.1M, 0.25M and 0.5M lithium chloride to 50 mg of dry cells expressing Escherichia coli PRT468, and conduct 30 minutes to process. After cooling to room temperature, centrifugation was performed at 11,000×g for 5 minutes. The supernatant (first soluble fraction) obtained by centrifugation was analyzed by SDS-PAGE. show the result in figure 1 . The numbers from 40 to 60 indicate the temperature, and the symbols from 0 to 0.5M indicate the concentration of lithium chloride contained in DMSO. Lane XL is the result of electrophoresis of molecular weight marker proteins, and lane C is the result of electrophoresis o...

Embodiment 2

[0256] [Example 2 One of the effects of adding cyclohexanone in the solubilization step of host cell-derived protein]

[0257] In Example 1, when DMSO containing a high concentration of lithium chloride was used as the first aprotic polar solvent, not only the dissolution of the host cell-derived protein but also the dissolution of the target protein PRT468 was confirmed. Therefore, the effect of preventing the dissolution of PRT468 by adding cyclohexanone to DMSO containing a high concentration of lithium chloride was investigated.

[0258] Prepare solvents with three mixing ratios of DMSO:cyclohexanone=100:0, 50:50, and 25:75, add lithium chloride to these solvents so as to reach 0.1M or 0.5M, and prepare a total of six kinds of first Aprotic polar solvents (see Table 5).

[0259] [table 5]

[0260] Lane No.

Lithium chloride (M)

DMSO(%)

Cyclohexanone(%)

1

0.1

100

0

2

0.1

50

50

3

0.1

25

75

4

0.5

...

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Abstract

Disclosed is a method for purifying a recombinant protein from a recombinant cell in which a target recombinant protein is expressed within the cell as an insoluble body, said method characterized inthat said recombinant cell is treated under conditions in which a protein derived from a host cell would dissolve, but said recombinant protein would not dissolve, in a first aprotic polar solvent towhich an inorganic salt has been added or not added, a first soluble fraction is removed, and then an obtained first insoluble fraction is treated under conditions in which the recombinant protein would dissolve in a second aprotic polar solvent to which an inorganic salt has been added.

Description

technical field [0001] The present invention relates to a method for purifying the recombinant protein from recombinant cells expressing the recombinant protein. Background technique [0002] The use of genetically recombined host cells enables the production of target proteins on an industrial scale. Many methods for isolating and purifying recombinant proteins produced by recombinant cells have also been reported. [0003] As a method for isolating an insoluble target protein, a method using metal hydroxides such as sodium hydroxide from a suspension of recombinant cells (Patent Document 1), and a method using organic acids such as formic acid and propionic acid (Patent Document 1) have been reported. 2) etc. However, when an insoluble target protein is isolated by these methods, it is difficult to remove impurities present with the target protein, so there is a problem that the isolated target protein is not high in purity. In addition, in the case of the method using ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K1/30C07K1/36C07K14/435C12P21/02D01F6/68
CPCD01F6/68C07K1/145C07K14/78C07K14/43518C07K14/43586C07K2319/21C07K1/303D01F4/00D01D1/02D01D5/06C07K1/36C07K1/30C07K14/435C12P21/02C07K1/14
Inventor 本间俊将
Owner SPIBER INC
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