Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2001-03-02
2003-05-27
Weber, Jon P. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S402000, C435S183000
Reexamination Certificate
active
06569999
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for activating a denatured protein. In particular, the present invention relates to a method for activating a denatured protein which includes the steps of: adding a detergent to a denatured protein to form a protein-detergent complex; and adding high-molecular weight amylose to the protein-detergent complex so that the high-molecular weight amylose removes the detergent. Moreover, the present invention relates to a protein refolding kit including at least one kind of high-molecular weight amylose and at least one kind of detergent.
2. Description of the Related Art
A protein includes a polypeptide chain consisting of a plurality of L-&agr;-amino acids which are linked via peptide bonds. The order in a sequence of amino acids of a protein is referred to as a primary structure. An actual protein assumes a three-dimensional conformation referred to as a “higher-order structure”. In order for a protein to function, it is essential that the protein assumes a proper higher-order structure. Higher-order structures of proteins include secondary structures, tertiary structures, and quaternary structures. Examples of secondary structures include the &agr; helix structure, &bgr; sheet structure, and the like. Such secondary structures may be further folded to give tertiary structures. Examples of bonds which make for the stabilization of tertiary structures include hydrophobic bonds, hydrogen bonds, and S—S bonds between cysteine residues. Through the non-covalent association of a plurality of polypeptide chains each assuming a tertiary structure, a quaternary structure results.
A structure which is inherently assumed by a protein molecule under substantially physiological conditions is referred to as a “native” structure. The term “denaturation” refers to the alteration of the physicochemincal properties of a protein from its native state, which may be induced by various factors which cause the destruction of a higher-order structure without causing changes in the primary structure. The destruction of a higher-order structure of a protein may occur due to physical causes, e.g., heating, freezing, irradiation by ultraviolet or X rays, as well as chemical causes, e.g., extremely alkaline conditions, denaturing agents such as organic solvents, urea, and guanidine hydrochloride, or detergents. As a result of denaturation, the structure of a protein may change from a compact structure with orderly folds to an irregular aggregated structure, or a random structure in which the folds have been unfolded. As used herein, a “denatured state” includes both irregularly aggregated states and unfolded states.
A so-called “refolding technique”, i.e., a technique for refolding a protein which is in a denatured state as defined above in such a manner as to restore a proper higher-order structure, has very important implications in the industrial utilization of proteins. For example, an enzyme which has been utilized for a certain reaction for producing a substance may be denatured (through heating or the addition of a denaturing agent), whereby its enzyme activity is lost so that the reaction stops; after the resultant product has been recovered, the denatured enzyme may be refolded so as to restore a proper higher-order structure, i.e., activated. Thus, such an enzyme can be put to further reuse.
When heterogeneous proteins are produced in an
E. coli
host, a number of proteins may often become insoluble and inactive substances, referred to as “inclusion bodies”. However, with a refolding ability, such an insoluble protein may be first solubilized by a denaturing agent such as urea or guanidine hydrochloride, and thereafter refolded so as to restore a proper higher-order structure, i.e., activated.
The technique of refolding a denatured protein for restoring a proper higher-order structure is associated with two specific problems to be solved: the first problem is how to prevent aggregation of protein; the second problem is how to properly refold the unfolded protein molecules back into a protein.
In the in-vivo folding of a protein, a class of assisting proteins, termed “molecular chaperons”, are known to be involved in the above two steps. Molecular chaperons are proteins which bind to a protein which has just been synthesized so that the protein is prevented from being folded in an irregular manner and can be readily transported, and/or assist in the folding process of proteins which would otherwise have difficulties in folding.
A “nascent protein” is a protein which has just been translated in vivo and has not assumed a proper higher-order structure. Immediately after the completion of translation, a nascent protein is bound by a class of molecular chaperons called DnaJ, etc. These molecular chaperons operate upstream of the folding process so as to prevent nascent protein from aggregating or assuming abnormal structures. Thereafter, another molecular chaperon called GroE, which operates downstream of the folding process, acts on the nascent protein. Owing to the action of GroE, the nascent protein gradually begins to assume a proper higher-order structure, until it is finally folded, into an active protein. In the course of the folding process, the molecular chaperons which assisted in the folding leave the nascent protein.
In recent years, several attempts have been made to construct artificial chaperons with a view to reproducing the in-vivo functions of molecular chaperons In vitro (e.g., in a test tube) and restoring the activity of a denatured protein in vitro. Daugherty et al. (J. Biol. Chem., 273,3961-33971 (1998)) reported a method of refolding a denatured protein by using artificial chaperons. The reported method employs a non-ionic detergent designated Triton X-100, and a polyoxyethylene-type detergent having a short alkyl group chain, as artificial chaperons which function to prevent aggregation of proteins. After a protein-detergent complex is formed by using these artificial chaperons, &bgr; cyclodextrin (hereinafter abbreviated as “&bgr; CD”), which is a low-molecular weight cyclic &agr;-1,4-glucan, is added as a substance (hereinafter also referred to as a “detergent removing agent”) for causing removal of the detergents. Thus, the detergents are gradually removed from the protein-detergent complex, thereby allowing the denatured protein to naturally assume a higher-order structure.
Thereafter, Silvakama Sundari et. al (FEBS Lett., 443, 215-219 (1999) ) discloses that, not only a low-molecular weight cyclic &agr;-1,4-glucan (cyclodextrin), but also a low-molecular weight straight-chain &agr;-1,4-glucan can be effective as a substance for causing gradual removal of detergents from a protein-detergent complex in a similar artificial chaperon system.
More recently, Machida et al. (Japanese Patent Application No. 2000-71533) is a study specifically into the effects of various combinations of detergents and detergent removing agents on the restoration of the activity of three different proteins in a similar artificial chaperon system, reporting the following results:
(1) High-molecular weight cyclic &agr;-1,4-glucan (having a polymerization degree of about 40 to about 150) enables faster and more effective restoration of protein activity than low-molecular weight cyclic &agr;-1,4-glucan (having a polymerization degree of about 6 to about 8).
(2) Each protein may have its activity restored to various degrees depending on the detergents and the detergent removing agents used. Therefore, for a higher level restoration of activity, it is essential to select appropriate combinations of detergents and detergent removing agents in accordance with the protein to be restored.
Thus, it has been indicated that the artificial chaperon technology is very effective for the activation of denatured proteins. On the other hand, it has also been learned that the degree of activity restoration of a denatured protein may substantially vary depending on the detergents and the detergent removing agents used.
A la
Fujii Kazutoshi
Hayashi Kiyoshi
Machida Sachiko
Takaha Takeshi
Terada Yoshinobu
Fay Sharpe Fagan Minnich & McKee LLP
National Food Research Institute, Ministry of Agriculture, Fores
Weber Jon P.
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