Protein refolding agent and refolding method

Abstract

A refolding agent and refolding method which makes it possible to produce high-purity proteins in high productivity. The refolding agent includes a phosphorus-containing compound (A) and an oxycarbonyl group-containing compound (B). The refolding method includes the step of treating the unfolded protein with the refolding agent. As the compound (A), there may be at least one selected from inorganic phosphoric acids, alkyl phosphate esters, sugar phosphate esters, and salts of these, and as the compound (B), there may be at least one selected from formic acid, acetic acid, propionic acid, lactic acid, tartaric acid, and salts of these.

Description

TECHNICAL FIELD[0001]The present invention relates to a protein refolding agent and a protein refolding method and, more particularly, to a refolding agent to be used for refolding an unfolded protein to an active protein structure, and a method of such refolding.BACKGROUND ART[0002]It is very important to elucidate and analyze the function and structure of a protein, since it may be directly connected with the treatment of a disease or the creation of a new drug, for instance. Therefore, intensive studies have been made for synthesizing or producing various proteins by various methods, examining the structures thereof, and elucidating the mechanisms of action and the roles thereof in living organisms. It is now well known that the function of a protein is decided not only by the sequence of amino acids constituting the protein and the chain length but also by an orderly three-dimensional structure (higher order structure) it exhibits.[0003]From the industrial viewpoint as well, the...

AI Technical Summary

Benefits of technology

[0149]When the refolding agent and refolding method according to the invention are used, the productivity is increased dramatically as compared with the prior art since no large scale protein dilution is required. In addition, since the refolding effect is higher than in the prior art, it is possible to obtain high-purity proteins in large amounts.

Problems solved by technology

However, many of the proteins expressed in recombinants are not orderly in three-dimensional structure or not controlled in higher order structure but often form inactive small-particle granules called inclusion bodies.

However, the methods are mostly low in refolding rate; in addition, in many cases, favorable results were obtained incidentally with certain limited proteins (in particular specific low-molecular-weight proteins).

However, the dialysis method is not practical from the industrial point of view since the required volume of the dialyzing fluid generally amounts to at least 100 times the volume of the protein solution and, further, a period of several days is required.

While the dilution method is a most simple and low-cost method of refolding a protein to its functional three-dimensional structure, the refolding yield rate is low (e.g. Non-Patent Document 2) and the high dilution ratio leads to a low yield under the existing circumstances.

However, this method cannot always give sufficiently high refolding rates; generally, no satisfactory results can be obtained with other proteins.

However, such refolding is observed only with a very limited number of proteins, for example cyclophilin A. In particular, under the present conditions, the use of a molecular chaperone, which is a certain kind of template, cannot serve at all when the protein to be refolded is incompatible with the template.

However, the situation is the same: this method is applicable only to such protein and the resin preparation is complicated and results in increased costs.

Furthermore, the method cannot be carried out repeatedly; hence, it is an expensive method.

In spite of such various proposed methods of refolding as discussed above, problems are still encountered in refolding unfolded proteins, namely low yields resulting from high-ratio dilution of the proteins as well as low purity levels.

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