Protein Refolding Method

a refolding method and protein technology, applied in the field of refolding methods, can solve the problems of protein loss, destabilization, and difficulty in refolding techniques for people without experience, and achieve the effects of preventing association and/or aggregation, and facilitating and effectively refolding a protein

Inactive Publication Date: 2014-11-27
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]Thus, it is an aspect of the present invention to provide a simple refolding method, which allows for the restoration of the protein's native higher-order structure while smoothly stripping away the surfactant from the protein.
[0027]According to the present invention, it is possible to easily and effectively refold a protein without any experience in refolding methods or using any special device. According to the present invention, it is also possible to prevent association and / or aggregation of a protein which may occur during the refolding process.
[0028]Although not bound by any theory, it is considered as follows. A protein in general associates and / or aggregates as soon as a surfactant is stripped from the protein. In the meantime, according to the present invention, when a denatured protein is unfolded by using a specific surfactant and then diluted, and arginine or an arginine derivative is added to the thus obtained diluted solution, it is possible to prevent association and / or aggregation of the protein by the arginine or arginine derivative while allowing the specific surfactant to be detached from the protein.

Problems solved by technology

Often, the protein has also lost its activity and / or become destabilized.
However, reagents and methods used in refolding must be appropriately selected for each protein; therefore, such techniques can be difficult for a person with no experience in refolding.
In addition, even for a person with sufficient experience, it still may be difficult to re-acquire the native state of a protein if the protein has a complicated higher-ordered structure and is likely to associate and / or aggregate during refolding.
However, as the artificial chaperone system was increasingly used, the following facts and the like were revealed: the stripping of the added surfactant from the protein is not as easy as reported; and complicated experiments are still required in order to determine the appropriate re-folding conditions.
In addition, the multi-stage operation complicates the process, and therefore limits the application to industrial-scale production (H. Lanckriet and A. P. J. Middelberg: Biotechnology Progress 20, 1861-1867 (2004)).
As described above, even the artificial chaperone system which has received the highest evaluation is not sufficient as a method which allows a person with no experience to easily and effectively carry out protein refolding.
In this method, it is difficult to effectively restore the native state of any protein other than the growth hormone.

Method used

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Examples

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Effect test

reference example 1

[0115]As a protein, human interleukin-6 (rhIL-6: Japanese Patent No. 3200850) prepared from a recombinant E. coli strain was used. The rhIL-6 is insoluble in water (solubility to 100 g of water at 25° C.: 0.001 g or less), and is in a granular form. Aliquots of the insoluble granules containing 4 mg of rhIL-6 were each put into an Eppendorf tube (made of polypropylene: available from Eppendorf Co., Ltd.). The amount of rhIL-6 in the insoluble granules was quantified in advance by the reverse-phase HPLC method described in a published report (Japanese Patent No. 3200850).

[0116]To each of these, a surfactant prepared in advance to be 5% in pure water (Milli Q water) were appropriately added to achieve a final concentration of lauroyl-L-Glu, lauroyl-L-Asp, lauroyliminodiacetic acid, decanoylSar, decanoyl-L-Ala, decanoic acid, or lauryltrimethylammonium chloride of 2% and the rhIL-6 extraction concentration of 4 mg / ml, and the final volume of 1 ml was adjusted to 1 ml. The mixture was i...

reference example 2

[0124]It was investigated whether lauroyl-L-Glu, lauroyl-L-Asp, and lauroyliminodiacetic acid were effective in restoration of the native higher-order structure of the insoluble rhIL-6, even if the concentration of rhIL-6 to be subjected to extraction from the insoluble granules and solubilization thereafter was increased. For comparison, the following surfactants were used: lauryltrimethylammonium chloride, which resulted in an amount of rhIL-6 of 80% or more as compared to that of lauroyl-L-Glu in Reference Example 1; lauroyl-Sar, which has the same lauroyl group; undecanoyl-L-Glu, tridecanoyl-L-Glu, and myristoyl-L-Glu, which were used for confirmation of the effect of the length of the acyl chain in lauroyl-L-Glu; and decanoic acid, which was used for confirmation of the effect of the acyl chain.

[0125]Aliquots of 13 mg of the same insoluble granules of rhIL-6 as used in Reference Example 1 were each put into an Eppendorf tube. The surfactants were added to the respective Eppendo...

reference example 3

[0128]The concentration of surfactant for extracting rhIL-6 from the insoluble granules for solubilization was investigated.

[0129]Aliquots of 12.6 mg of the same water-insoluble granules of rhIL-6 as used in reference Example 1 were each put into an Eppendorf tube. A solution of lauroyl-L-Glu was added to the Eppendorf tubes to respectively achieve final concentrations of Lauroyl-L-Glu of 1.5%, 1.75%, 2.00%, 2.25%, and 3.00%, and thereby 3 ml of each solution of 10 mM sodium phosphate, pH 7.0 (25° C.) were added. The solutions were incubated at room temperature for 2 hours, thereby extracting rhIL-6 from the insoluble granules and solubilizing rhIL-6. If rhIL-6 was completely extracted, the concentration of the extraction of rhIL-6 would be adjusted to 4.3 mg / ml as a result.

[0130]After the extraction, 10 mM dithiothreitol (DTT) was added to a supernatant, and the supernatant containing DTT was adjusted to pH 8 and then heated at 37° C. for 30 minutes to reduce the disulfide bond. Th...

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Abstract

The present invention provides a method for producing a protein which has a restored native higher-order structure by bringing a protein which has lost its native higher-order structure into contact at pH 6.5 to 9.0 with a 1 to 3% aqueous solution of a specific surfactant, such as lauroylglutamic acid to obtain a solubilized solution of the protein; and then adding the solubilized solution to a buffer with pH 6.5 to 9.0 containing arginine or an arginine derivative at a concentration of 0.1 to 1.2 M to lower the concentration of the specific surfactant, such as lauroylglutamic acid, in the obtained mixture solution down to 0.02 to 0.275%. According to the present invention, it is possible to easily restore the native higher-order structure of a protein while smoothly removing the surfactant from the protein.

Description

[0001]The application is a continuation of, and claims priority under 35 U.S.C. §120 to, U.S. patent application Ser. No. 12 / 941,272, filed on Nov. 8, 2010, which was a continuation under 35 U.S.C. §120 to International Application No. PCT / JP2009 / 058304, and claims priority therethrough under 35 U.S.C. §§119, 365 to Japanese Application No. 2008-122536, the entireties of which are hereby incorporated by reference herein.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to a refolding method for restoring the native higher-order structure of a protein which has lost activity and / or stability as a result of becoming insoluble or losing its higher-order structure.[0004]2. Brief Description of the Related Art[0005]When preparing a recombinant protein using a production host, such as E. coli, the protein can become denatured and / or insoluble in water, also known as denatured. Often, the protein has also lost its activity and / or become destabili...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/54C07K16/44C07K16/18C12N9/10
CPCC07K14/5412C12N9/1044C07K2317/569C07K16/18C07K2317/64C07K16/44C07K1/1136C07K1/145C07K14/475C12N9/104
Inventor YUMIOKA, RYOSUKEEJIMA, DAISUKE
Owner AJINOMOTO CO INC
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