JP2006111572A - Chaperonin-protein complex, method for producing the same, and method for producing the target protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chaperonin-protein complex that can be easily produced and can maintain a complex structure more stable than a natural one.
In the chaperonin-protein complex formed by assembling a plurality of chaperonin subunits and a target protein, a part of the plurality of chaperonin subunits and a target protein form a fusion protein, and the fusion protein Is a combination of one chaperonin subunit or chaperonin subunit conjugate and the target protein via a peptide bond, and the remainder of the plurality of chaperonin subunits is a peptide bond with the target protein and other chaperonin subunits. A chaperonin-protein complex, which is a single chaperonin subunit that is not linked via a non-covalent association of the fusion protein and the single chaperonin subunit.
[Selection] Figure 1

Description

  The present invention relates to a chaperonin-protein complex, a method for producing the same, and a method for producing a target protein. More specifically, a fusion protein of a chaperonin subunit and a target protein and a single chaperonin subunit are noncovalently bonded. The present invention relates to an associated chaperonin-protein complex, a method for producing the same, and a method for producing a target protein using the chaperonin-protein complex.

  A technique for mass-producing a target protein using a recombinant is one of basic techniques in the biotechnology field. In this technique, microorganisms such as bacteria and yeast, or cells derived from higher organisms such as animal cells and plant cells are used as host cells. Among these, recombinants using bacteria as host cells are favorably used for industrial-scale protein production because their growth rate is markedly greater than that of animal cells and culturing is easy.

  However, the following problems are always accompanied when the target protein is produced by a recombinant. One example is when the target protein is toxic to the host cell. In this case, gene expression of the target protein may be suppressed or proliferation of the recombinant may be inhibited, and as a result, the target protein may be hardly obtained. In another example, the synthesized target protein may be degraded by the attack of a host cell-derived protease, resulting in almost no target protein being obtained. In yet another example, the synthesized target protein may not be correctly folded in the host cell, and may only be obtained as an abnormal protein that does not have the correct three-dimensional structure. In particular, when the recombinant protein is not correctly folded, insoluble aggregates (inclusion bodies) are often formed. And, it is difficult to refold the abnormal protein that has once become an inclusion body to return it to the correct three-dimensional structure, and it is often unsuccessful.

  As a method for solving the above problems, a method using chaperonin, which is a kind of molecular chaperone, is known. Chaperonin is a type of molecular chaperone that is abundant in bacterial cytoplasm, eukaryotic mitochondria, and chloroplasts, and has the activity of promoting protein folding and blocking protein denaturation. . Many of chaperonins are known to be heat shock proteins (heat shock proteins) induced by stress such as heat shock.

  Chaperonin is a large cylinder-like complex protein having a total molecular weight of about 800,000 to 1,000,000, in which two ring-shaped structures composed of 7 to 9 subunits having a molecular weight of about 60,000 (chaperonin subunits) overlap. The chaperonin has a cavity inside the ring-shaped structure, and a protein (hereinafter referred to as “chaperonin-protein complex”) in which the protein being folded or denatured is temporarily stored in the cavity. ). And it is known that the protein accommodated in the cavity is correctly folded, and then the correctly folded protein is released from the cavity.

Taking GroEL, which is a chaperonin derived from Escherichia coli as an example, GroEL has a structure in which two ring-shaped structures consisting of seven GroEL subunits are overlapped, and is formed from a total of 14 GroEL subunits. (FIG. 10). The cavity inside the GroEL ring-shaped structure has an inner diameter of 4.5 nm and a height of 14.6 nm, which is just large enough to accommodate a 60 kDa globular protein. GroEL can accommodate various proteins in its cavity to form a chaperonin-protein complex. And the protein stored in the cavity is correctly folded and then released from the cavity.

  There is an attempt to obtain a normal protein having a correct three-dimensional structure by correctly folding the target protein by utilizing such action of chaperonin. That is, the target protein and chaperonin are contacted to form a chaperonin-protein complex, the target protein is correctly folded by the action of the chaperonin, and the target protein is obtained as a normal protein having a correct three-dimensional structure. For example, chaperonin and the target protein may be co-expressed in the same host, and the target protein may be obtained as a normal protein.

  This chaperonin-protein complex has an action of protecting the protein from protease attack in addition to the action of correctly folding the stored protein. Furthermore, even if the stored protein is toxic to the host cell, its toxicity can be suppressed if it is in the form of a chaperonin-protein complex. These phenomena are thought to be because in the chaperonin-protein complex, the protein is contained in the cavity of the chaperonin and is not exposed to the outside.

  The chaperonin-protein complex described above exists in nature, but a chaperonin-protein complex can also be formed by a fusion protein of a chaperonin and a target protein. For example, Patent Document 1 and Patent Document 2 obtain a fusion protein in which a GroEL subunit and a target protein are linked at a number ratio of (7: 1), (4: 1) or (7: 2), It is described that a chaperonin-protein complex was formed only by these fusion proteins.

JP 2004-24095 A JP 2004-81199 A

  However, natural chaperonins may not be able to stably store proteins in cavities depending on the structure and molecular weight of the target protein. For example, it is said that the ratio of proteins that can be stored in natural chaperonin out of all proteins present in E. coli is only 20-30%.

On the other hand, when a chaperonin-protein complex is formed only with the fusion protein, the fusion protein does not exist in nature, and therefore, it is necessary to construct a fusion gene encoding the fusion protein. Then, it is necessary to incorporate the fusion gene into an appropriate expression vector, introduce the expression vector into an appropriate host, and express the fusion gene in the host. At this time, the larger the size of the fusion gene to be incorporated, the larger the size of the expression vector. In general, however, the larger the size of an expression vector such as a plasmid, the more unstable the host will cause various problems. There is. For example, replication of the expression vector itself may not be performed successfully, or DNA that has been integrated through homologous recombination may be lost, resulting in insufficient production of the chaperonin-protein complex. Taking the case where the host cell is Escherichia coli as an example, the upper limit of the size of the plasmid stably retained in the Escherichia coli cell is about 100,000 base pairs. However, since the molecular weight of the above-mentioned fusion protein is the molecular weight of a plurality of chaperonin subunits and the target protein, the size of the fusion gene encoding it is large, and the size of the plasmid vector incorporating the fusion gene is 100,000. Much more than base pairs. Therefore, holding a plasmid vector in which the fusion gene is incorporated in E. coli still has problems in terms of the stability of the plasmid vector, which may hinder the productivity of the chaperonin-protein complex.

  From the above, a chaperonin-protein complex that can stably store a target protein in a cavity and is also excellent in productivity is desired.

  The invention according to claim 1 for solving the above-mentioned problem is a chaperonin-protein complex in which a plurality of chaperonin subunits and a target protein are aggregated, and a part of the plurality of chaperonin subunits and a target protein. Form a fusion protein, wherein the fusion protein comprises one chaperonin subunit or chaperonin subunit conjugate and the target protein linked via a peptide bond, and the plurality of chaperonin subunits The remainder is a single chaperonin subunit that is not linked to the target protein and other chaperonin subunits via peptide bonds, and the fusion protein and the single chaperonin subunit are associated non-covalently. Chaperoni featuring - it is a protein complex.

  The chaperonin-protein complex of the present invention is formed by aggregating a plurality of chaperonin subunits and a target protein, and one chaperonin subunit or a chaperonin subunit conjugate and a target protein are bonded via peptide bonds. The linked fusion protein and a single chaperonin subunit are associated non-covalently. Among these, in the fusion protein, the target protein and chaperonin subunits and between each chaperonin subunit (when there are a plurality of chaperonin subunits) are firmly linked by covalent bonds. On the other hand, a single chaperonin subunit is not covalently linked to the fusion protein or to another single chaperonin subunit. That is, in the chaperonin-protein complex of the present invention, the target protein and a plurality of chaperonin subunits are assembled in a manner in which a covalent bond and a noncovalent bond are mixed to form a complex. With such a configuration, the molecular weight of the fusion protein is suppressed to some extent and its gene expression is facilitated, that is, it is easy to produce, while maintaining a more stable complex structure than the natural chaperonin-protein complex. .

  Here, the term chaperonin subunit is used not only when it is a single molecule (single chaperonin subunit) but also when it is part of a fusion protein. The chaperonin subunit linked body refers to an artificial protein in which two or more chaperonin subunits are linked in tandem. Further, a chaperonin subunit linked body composed of N chaperonin subunits is called a chaperonin subunit N-fold linked body (however, when N = 1, it means one chaperonin subunit) .) Furthermore, “non-covalent” refers to a bond mode other than a covalent bond, such as hydrogen bond, hydrophobic interaction, ionic bond, van der Waals force and the like. In this specification, the term “bond” is used only for a covalent bond, and the term “association” is used for a non-covalent bond.

The number of target proteins constituting the chaperonin-protein complex of the present invention may be one or plural. Furthermore, when there are a plurality of target proteins, the plurality of target proteins may be included in the same fusion protein, or may be included in different fusion proteins. In other words, the fusion protein constituting the chaperonin-protein complex of the present invention includes one or a plurality of target proteins. Further, the binding site of the target protein contained in the fusion protein may be either the N-terminal side or the C-terminal side of one chaperonin subunit or chaperonin subunit conjugate. Furthermore, it is also possible to bind between the chaperonin subunits of the chaperonin subunit conjugate. When the fusion protein includes a plurality of target proteins, a plurality of binding sites can be selected from among the above C-terminal, N-terminal, and chaperonin subunits. In addition, the target protein may be directly linked to the above binding site, or indirectly linked via a linker peptide or the like.

  The invention according to claim 2 is the chaperonin-protein complex according to claim 1, wherein the number of the target proteins is one.

  In order for chaperonin to form a complex with the target protein, there is an upper limit on the molecular weight of the protein. That is, a protein having a too large molecular weight cannot form a complex with chaperonin. In addition, in order for a plurality of proteins to form a complex with chaperonin, the total molecular weight of the proteins needs to be not more than the upper limit value. That is, in this case, the molecular weight of each protein needs to be considerably smaller than the upper limit. In the chaperonin-protein complex of the present invention, the number of proteins is one. With this configuration, a large protein whose molecular weight is approximately the same as the upper limit value can be included.

  The invention according to claim 3 is characterized in that the fusion protein is one in which one chaperonin subunit or a chaperonin subunit linked body and a target protein are linked via a linker peptide. 3. The chaperonin-protein complex according to 1 or 2.

  In the chaperonin-protein complex of the present invention, the target protein is linked to one chaperonin subunit or a chaperonin subunit linked body via a linker peptide. That is, by selecting a linker peptide having a desired function, the chaperonin-protein complex of the present invention can have a new function.

  The invention according to claim 4 is the chaperonin-protein complex according to claim 3, wherein the linker peptide includes a recognition site for a limited-degradation protease.

  The invention according to claim 5 is the chaperonin-protein complex according to claim 4, wherein the limited-degradation protease is thrombin.

  In the chaperonin-protein complex of the present invention, the linker peptide contains a recognition site for limited-degradation protease. With such a configuration, the recognition protein is hydrolyzed and cleaved, and the target protein is cut out by allowing a limited-degradation protease for the recognition site to act on the fusion protein released from the chaperonin-protein complex of the present invention. It becomes possible.

  The invention according to claim 6 is the chaperonin-protein complex according to claim 4 or 5, wherein the recognition site is not cleaved by the action of the limited degradation protease.

In the chaperonin-protein complex of the present invention, the recognition site contained in the linker peptide is not cleaved by the action of the limited degradation protease. The fact that the recognition site is not cleaved by a protease means that the recognition site is not exposed to the outside in the chaperonin-protein complex of the present invention, that is, the target protein is protected by being surrounded by a plurality of chaperonin subunits. Related to that. With such a configuration, in the chaperonin-protein complex of the present invention, the target protein can be folded more reliably and can be obtained as a normal protein having a correct three-dimensional structure. When the target protein is excised from the chaperonin-protein complex of the present invention, the chaperonin-protein complex of the present invention is dissociated into a fusion protein and a single chaperonin subunit to release the fusion protein. It is necessary to allow limited degradation protease to act on the fusion protein.

  The invention according to claim 7 is the chaperonin-protein complex according to any one of claims 1 to 6, wherein the plurality of chaperonin subunits are subunits of a group 1 type chaperonin.

Chaperonins are classified into group 1 type chaperonins that require a cofactor for their activity expression and group 2 type chaperonins that do not require a cofactor and can be independently expressed. The chaperonin-protein complex of the present invention comprises the chaperonin subunit of group 1 type chaperonin as a constituent element, and correctly folds the target protein with the help of a cofactor. In the chaperonin-protein complex of the present invention, all of the plurality of group 1 type 1 chaperonin subunits may be derived from the same species of organism, or may be derived from different organisms.

  The invention according to claim 8 is the chaperonin-protein complex according to claim 7, wherein all of the subunits of the group 1 type chaperonin are derived from the same organism.

  In the chaperonin-protein complex of the present invention, the chaperonin subunit is derived from the same organism. With this configuration, a complex is more reliably formed and the complex structure is stable.

  The invention according to claim 9 is the chaperonin-protein complex according to claim 7, wherein all subunits of the group 1 type chaperonin are derived from E. coli.

  A chaperonin derived from E. coli is a group 1 type chaperonin, and its biochemical and physicochemical properties are well studied. Moreover, the gene of the subunit has also been isolated. Therefore, a fusion gene encoding the fusion protein can be easily constructed. With this configuration, the chaperonin-protein complex of the present invention is easy to produce, and the target protein can be obtained as a normal protein more easily.

  In addition, the invention according to claim 10 is characterized in that the total number of the plurality of chaperonin subunits is an optimum number determined by the origin of the chaperonin subunits. The chaperonin-protein complex.

  In nature, the total number of chaperonin subunits forming chaperonin is largely determined by the origin of chaperonin. For example, the number is 7 for bacteria, 8 or 9 for archaebacteria, and 8 for eukaryotes. That is, these numbers are the optimal numbers for each chaperonin. In the chaperonin-protein complex of the present invention, the total number of chaperonin subunits is the optimum number determined by the origin of the chaperonin subunits. Therefore, in the chaperonin-protein complex of the present invention, the complex structure is stable.

  The invention according to claim 11 is the chaperonin-protein complex according to claim 10, wherein the optimal number is seven.

In the chaperonin-protein complex of the present invention, the total number of chaperonin subunits is seven. With this configuration, when the chaperonin subunit is derived from bacteria, the complex structure is stable. In the chaperonin-protein complex of the present invention, the sum of the number of chaperonin subunits contained in the fusion protein and the number of single chaperonin subunits is 7. Therefore, the number of chaperonin subunits contained in the fusion protein and the number of single chaperonin subunits both range from 1 to 6.

  The invention according to claim 12 is characterized in that the plurality of chaperonin subunits form a ring-shaped structure having a cavity therein, and the target protein is stored in the cavity. 12. A chaperonin-protein complex according to any one of 11 to 11.

  In natural chaperonins, a plurality of chaperonin subunits form a ring-shaped structure, and it is assumed that there is a cavity inside the ring-shaped structure. Then, another protein is stored in the cavity to form a chaperonin-protein complex. The chaperonin-protein complex of the present invention has a structure similar to that of a natural chaperonin-protein complex. That is, a ring-shaped structure is formed, and there is a cavity inside, and the target protein is stored in the cavity. According to the chaperonin-protein complex of the present invention, the target protein is correctly folded in the cavity inside the ring-shaped structure. Furthermore, in the chaperonin-protein complex of the present invention, since the target protein is stored in the cavity inside the ring-shaped structure, the target protein is not exposed to the outside. With this configuration, the target protein is not easily digested by proteases from the outside, and the target protein can be obtained in a higher yield.

  The invention according to claim 13 is characterized in that the ring-shaped structure formed by another chaperonin subunit is associated non-covalently with the ring-shaped structure. The chaperonin-protein complex described in 1.

  In nature, chaperonins often have a structure (double ring structure) in which two ring-shaped structures are associated non-covalently. One of the two ring-shaped structures is distinguished as a cis ring that stores the target protein, and the other as a trans ring, and forms a chaperonin-protein complex in the cis ring. In the chaperonin-protein complex of the present invention, the ring-shaped structures formed by other chaperonin subunits are associated non-covalently and form a double ring structure as a whole. With this configuration, the chaperonin-protein complex of the present invention functions as a cis ring, and the target protein stored in the cavity can be correctly folded. In addition, in the ring-shaped structure formed by another chaperonin subunit, the inside may remain a cavity, and the target protein may be stored inside. When the inside remains hollow, the ring-shaped structure functions as a trans ring in the same manner as natural chaperonin. On the other hand, when the target protein is stored inside, the target protein is folded in both ring structures.

  The invention according to claim 14 is the chaperonin-protein complex according to any one of claims 1 to 13, wherein the target protein is a transmembrane receptor.

  The invention according to claim 15 is the chaperonin-protein complex according to claim 14, wherein the transmembrane receptor is a G protein-coupled receptor.

  The invention according to claim 16 is the chaperonin-protein complex according to claim 15, wherein the G protein-coupled receptor is a human endothelin A receptor.

  The transmembrane receptor is highly hydrophobic and can be obtained only in a state penetrating the cell membrane or insolubilized. However, according to the chaperonin-protein complex of the present invention, the transmembrane receptor can be obtained in a solubilized state, and can be used for applications such as drug development using the transmembrane receptor. Become. An example of a transmembrane receptor is a G protein-coupled receptor, and an example of a G protein-coupled receptor is a human endothelin A receptor.

  The invention according to claim 17 is a method for producing a chaperonin-protein complex comprising a plurality of chaperonin subunits and a target protein, and a gene encoding one chaperonin subunit or a chaperonin subunit conjugate. Transcribes and translates a fusion gene in which a gene encoding the target protein is linked and synthesizes a fusion protein in which one chaperonin subunit or chaperonin subunit linked body and the target protein are linked via a peptide bond And a method for producing a chaperonin-protein complex, wherein the fusion protein and a single chaperonin subunit are associated non-covalently.

  The method for producing a chaperonin-protein complex of the present invention is a method for producing a chaperonin-protein complex in which a plurality of chaperonin subunits and a target protein are aggregated, and includes one chaperonin subunit or a chaperonin subunit conjugate. A non-covalent association of a fusion protein in which a target protein and a target protein are linked via a peptide bond and a single chaperonin subunit. The protein is synthesized by transcription and translation of a fusion gene in which a gene encoding one chaperonin subunit or a chaperonin subunit conjugate and a gene encoding a target protein are linked. The system for expressing the fusion gene may be a system using a host or a cell-free translation system.

  The invention according to claim 18 is characterized in that a recombinant having the fusion gene is cultured, the fusion gene is transcribed and translated in the recombinant, and the chaperonin-protein complex is obtained from the recombinant culture. The method for producing a chaperonin-protein complex according to claim 17, wherein:

  The method for producing a chaperonin-protein complex of the present invention is carried out by a system using a host. That is, a recombinant having a fusion gene encoding a fusion protein is cultured, the fusion gene is transcribed and translated in the recombinant, and the chaperonin-protein complex is obtained from the recombinant culture. And According to the method for producing a chaperonin-protein complex of the present invention, since it is produced by culturing a recombinant, it is easy to scale up and mass production is possible. The recombinant can be produced, for example, by incorporating the fusion gene into an appropriate expression vector and introducing it into an appropriate host. A single chaperonin subunit may be expressed by incorporating a gene encoding the chaperonin subunit into a suitable vector and introducing it into the host. It may be expressed from a gene on genomic DNA.

  The invention according to claim 19 is the method for producing a chaperonin-protein complex according to claim 18, wherein the fusion gene is incorporated into an expression vector.

  In the method for producing a chaperonin-protein complex of the present invention, a fusion gene is incorporated into an expression vector, and is transcribed and translated. With this configuration, the expression of the fusion gene can be easily controlled.

  The invention according to claim 20 is characterized in that a single chaperonin subunit is synthesized by transcription and translation of a gene encoding a chaperonin subunit present on the genomic DNA of the recombinant. The method for producing a chaperonin-protein complex according to claim 18 or 19.

  In the method for producing a chaperonin-protein complex of the present invention, a single chaperonin subunit is synthesized by transcription and translation of a gene encoding a chaperonin subunit present on the genomic DNA of the recombinant. That is, in the method for producing a chaperonin-protein complex of the present invention, a single chaperonin subunit is supplied from the genomic DNA of the host. As a result, the host only needs to accept the fusion gene as a foreign gene, and the burden on the recombinant is small. With this configuration, the recombinant can be cultured more stably, and a chaperonin-protein complex can be produced in high yield.

  The invention according to claim 21 is characterized in that a single chaperonin subunit is synthesized by transcription and translation of a gene encoding a chaperonin subunit incorporated into an expression vector. 19. A method for producing a chaperonin-protein complex according to 19.

  In the method for producing a chaperonin-protein complex of the present invention, a single chaperonin subunit is synthesized by transcription and translation of a gene encoding a chaperonin subunit incorporated into an expression vector. With this configuration, the expression of a gene encoding a single chaperonin subunit can be easily controlled.

  The invention according to claim 22 is the method for producing a chaperonin-protein complex according to any one of claims 18 to 21, wherein the recombinant is Escherichia coli.

  In the method for producing a chaperonin-protein complex of the present invention, since the recombinant is Escherichia coli, the culture can be performed easily and at low cost.

In the invention described in claim 23, the target protein for excising and isolating the target protein from a fusion protein in which one chaperonin subunit or a chaperonin subunit conjugate and the target protein are linked via a peptide bond. In the manufacturing method,
(1) non-covalently associating the fusion protein with a single chaperonin subunit to form a chaperonin-protein complex;
(2) dissociating the chaperonin-protein complex formed in step (1) into a fusion protein and a single chaperonin subunit, and releasing the fusion protein;
(3) A step of cutting out and isolating the target protein from the fusion protein released in step (2),
A method for producing a target protein.

In the method for producing a target protein of the present invention, when a target protein is cut out and isolated from a fusion protein in which one chaperonin subunit or a chaperonin subunit conjugate and a target protein are linked via a peptide bond. First, the fusion protein and a single chaperonin subunit are associated to form a chaperonin-protein complex. The chaperonin-protein complex is then dissociated into a fusion protein and a single chaperonin subunit to release the fusion protein. Finally, the target protein is cut out and isolated from the released fusion protein. With this configuration, even if the target protein has properties such as being easily insolubilized due to its high hydrophobicity, it can be obtained as a correctly folded normal protein.

  In the invention of claim 24, in the step (1), a recombinant having a fusion gene encoding the fusion protein is cultured, the fusion gene is transcribed and translated in the recombinant, and chaperonin- 24. The method for producing a target protein according to claim 23, wherein a protein complex is formed.

  The invention according to claim 25 is the method for producing a target protein according to claim 24, wherein the recombinant is Escherichia coli.

  In the method for producing a target protein of the present invention, a chaperonin-protein complex is formed by culturing a recombinant such as Escherichia coli. With this configuration, even if the target protein is toxic to the host cell or is susceptible to protease attack, it can be produced without problems.

  The invention according to claim 26 is characterized in that in step (2), the chaperonin-protein complex is dissociated by the action of a denaturing agent, and the target protein according to any one of claims 23 to 25 is produced. Is the method.

  In the target protein production method of the present invention, the chaperonin-protein complex is dissociated by the action of a denaturing agent. Examples of the denaturing agent include urea and guanidine hydrochloride.

  In the invention according to claim 27, the fusion protein is obtained by binding one chaperonin subunit or a chaperonin subunit conjugate and a target protein through a recognition site of a limited-degraded protease, 27. The method for producing a target protein according to any one of claims 23 to 26, wherein in 3), the recognition site is cleaved by the action of the protease, and the target protein is cut out and isolated from the fusion protein. .

  In the method for producing a target protein of the present invention, the fusion protein constituting the chaperonin-protein complex has a recognition site for a limited-degradation protease. And the recognition site | part of this limited degradation protease is located between one chaperonin subunit or a chaperonin subunit coupling | bonding body, and a target protein. In the protein production method of the present invention, the recognition site is hydrolyzed and cleaved by allowing the corresponding limited degradation protease to act on the liberated fusion protein in step (3). The target protein contained in is cut out. The method for producing a protein of the present invention is simple because a target protein can be cut out only by treatment with a protease.

  The chaperonin-protein complex of the present invention is easy to produce and can maintain a more stable complex structure than the natural chaperonin-protein complex. Furthermore, according to the chaperonin-protein complex of the present invention, even a target protein that forms an inclusion body without being correctly folded when expressed alone in a recombinant body can be correctly folded. Furthermore, according to the chaperonin-protein complex of the present invention, even when the target protein is toxic to the host cell or susceptible to protease attack, it is stably maintained in the host cell.

According to the method for producing a chaperonin-protein complex of the present invention, a chaperonin-protein complex having a stable complex structure can be produced more easily.

  According to the method for producing a target protein of the present invention, even when the target protein easily forms inclusion bodies or has a high hydrophobicity, it can be obtained as a correctly folded normal protein. Furthermore, even when a recombinant is used, a target protein that is toxic to the host cell or a target protein that is susceptible to protease attack can be produced.

  Hereinafter, the best mode for carrying out the present invention will be described in detail.

In the chaperonin-protein complex of the present invention, one chaperonin subunit or a fusion protein in which a chaperonin subunit conjugate and a target protein are linked via a peptide bond and a single chaperonin subunit are non-covalently bound. Meeting. A structural example of the chaperonin-protein complex of the present invention will be specifically described with reference to a schematic diagram, taking as an example the case where the total number of chaperonin subunits is seven. First, the examples shown in FIGS. 1 (a) to 1 (f) are chaperonin subunits N-linked (N = 1 to 6) (N = 1 indicates one chaperonin subunit). It is a chaperonin-protein complex having a fusion protein in which one target protein is linked to the C-terminus or N-terminus. In the figure, white circles represent chaperonin subunits, white squares represent target proteins, and solid lines represent peptide bonds. That is, the chaperonin-protein complex shown in FIG. 1A is formed from a fusion protein of one chaperonin subunit and one target protein, and six single chaperonin subunits. The chaperonin-protein complex shown in FIG. 1 (b) is formed from a fusion protein of a double-ligated chaperonin subunit and one target protein, and five single chaperonin subunits. The chaperonin-protein complex shown in FIG. 1 (c) is formed from a fusion protein of a three-folded chaperonin subunit and one target protein, and four single chaperonin subunits. The chaperonin-protein complex shown in FIG. 1 (d) is formed from a fusion protein of a 4-fold chaperonin subunit and one target protein, and three single chaperonin subunits. The chaperonin-protein complex shown in FIG. 1 (e) is formed from a fusion protein of a 5-fold chaperonin subunit and a target protein, and two single chaperonin subunits. The chaperonin-protein complex shown in FIG. 1 (f) is formed from a fusion protein of a six-folded chaperonin subunit and one target protein, and one single chaperonin subunit.

When the target protein is linked to the chaperonin subunit N-linked product (N = 2 to 6), the binding site may exist between the chaperonin subunits. Such an example is shown in FIG. That is, the chaperonin-protein complex shown in FIG. 2 is formed from a fusion protein of a 5-fold chaperonin subunit and a target protein, and two single chaperonin subunits. Proteins are linked between chaperonin subunits.

In the above example, the case where the number of target proteins is one is shown, but the number of target proteins may be plural. As such an example, an example in which the number of target proteins is two is shown in FIGS. 3 (a) and 3 (b). In the figure, white squares and black squares are two target proteins that are different or the same. That is, the chaperonin-protein complex shown in FIG. 3 (a) is formed from a fusion protein in which each target protein is linked to both ends of a 4-fold chaperonin subunit and three single chaperonin subunits. ing. On the other hand, the chaperonin-protein complex shown in FIG. 3 (b) is composed of a fusion protein in which one chaperonin subunit and one target protein are linked, a chaperonin subunit twice-linked complex and another one. It is composed of a fusion protein to which a target protein is linked and four single chaperonin subunits. The above-described chaperonin-protein complex having a plurality of target proteins is suitable when the target protein is originally a protein having a quaternary structure as a whole. For example, the target protein is an immunoglobulin heavy chain. The case of a light chain is mentioned.

  In the above examples, the types of chaperonin subunits are all the same. However, in the chaperonin-protein complex of the present invention, different types of chaperonin subunits are mixed as long as the combination can form a complex. Also good. Such an example is shown in FIGS. 4 (a) and 4 (b). In the figure, white circles and black circles are two different chaperonin subunits, and the others are the same as in FIG. That is, in the chaperonin-protein complex shown in FIG. 4A, the chaperonin subunit constituting the fusion protein and the single chaperonin subunit are different types. Such a chaperonin-protein complex is, for example, a chaperonin-protein complex produced by a recombinant, in which the chaperonin subunits constituting the fusion protein are those other than those derived from the host, and a single chaperonin subunit is the host. And those supplemented by transcription / translation from the genomic DNA. In addition, the chaperonin-protein complex shown in FIG. 4 (b) includes two different chaperonin subunits in the fusion protein, and two types of single chaperonin subunits. The combination of different types of chaperonin subunits that can form a complex in this way is preferably selected from a plurality of types of chaperonin subunits belonging to the same group (group 1 or group 2). Examples of chaperonin subunits belonging to the same group include a combination of E. coli and therpermus chaperonin subunits (both group 1). For example, when E. coli is used as the host and the chaperonin subunit in the fusion protein is derived from a Thermus genus, the single chaperonin subunit is derived from E. coli, but a chaperonin-protein complex can be formed. The chaperonin subunits of different types are typically derived from different sources as described above, but also include the case of mutants modified by deletion or substitution of amino acid residues.

  The chaperonin-protein complex shown in FIGS. 1 to 4 has a total number of chaperonin subunits of 7, but the same configuration is possible even when the total number is other than 7. is there. For example, if it is a chaperonin subunit derived from archaea, the same structure is possible with a total number of 8-9.

In one preferred embodiment of the chaperonin-protein complex of the present invention, the chaperonin subunit or chaperonin subunit conjugate and the target protein are linked via a linker peptide. At this time, if a linker peptide having a specific function is selected, the chaperonin-protein complex also has a specific function. The preferred specific function of the linker peptide is typically the function of cleaving peptide bonds, but the recognition site for the limited-degradation protease is particularly preferred. Taking the case where the limited degradation protease is thrombin as an example, thrombin specifically recognizes the site having the amino acid sequence shown in SEQ ID NO: 9 and digests the site. Therefore, by designing a linker peptide containing the amino acid sequence shown in SEQ ID NO: 9, and designing a fusion protein in which the chaperonin subunit and the target protein are linked by peptide bonds via the linker peptide, the chaperonin of this embodiment is designed. -The protein complex acquires the function of cleaving the target protein by the action of thrombin. Examples of other limited degradation proteases include blood coagulation factor Xa, enterokinase, plasmin, chymotrypsin, precision protease, collagenase and the like. In addition to proteases, cyanogen bromide, hydroxylamine, and NBS skatole can also cleave specific amino acid sequences, so that these recognition sites can be included in the linker peptide. A self-cleaving peptide such as intein can also be included in the linker peptide. In this case, it is not necessary to make protease or the like act from the outside, and the linker peptide is cleaved autocatalytically.

  In another preferred embodiment of the chaperonin-protein complex of the present invention, a plurality of chaperonin subunits form a ring-shaped structure having a cavity therein, and the target protein is stored in the cavity. Further, it is particularly preferable that a ring-shaped structure formed by another chaperonin subunit is associated with the ring-shaped structure in a non-covalent manner.

In the ring-shaped structure formed by the chaperonin-protein complex of the present embodiment, the structure can be confirmed by observation with an electron microscope, for example. FIG. 5 shows an observation example using an electron microscope. That is, the chaperonin-protein complex shown in FIG. 5 has a ring-shaped structure in which chaperonin subunits are arranged concentrically. Moreover, when the chaperonin-protein complex of the present embodiment having a ring-shaped structure is subjected to gel filtration, the behavior resulting from the unique structure is exhibited. That is, when TSK-GEL G4000SW (Tosoh Corporation. Exclusion limit 7 × 10 ⁶ ) is used as a carrier, it is subjected to gel filtration chromatography at a flow rate of 0.5 mL / min and 25 ° C. to have a ring-shaped structure. The chaperonin-protein complex is eluted at a position with a residence time of around 17 minutes (elution volume 8.6 mL, 0.16 column volume (CV)) (Weissman, JS et al. Cell 84, 481-490). (1996)). Furthermore, when the chaperonin-protein complex of this embodiment having a ring-shaped structure is subjected to Native-PAGE, the behavior due to its unique structure is exhibited. That is, it is possible to detect a chaperonin-protein complex having a ring-shaped structure by subjecting it to Native-PAGE using a buffer solution having a pH of 8.8 by the Davis method at a gel concentration of 3% (Mendoza, JA). Et al. Biochim. Biophys. Acta. 1247, 209-214 (1995)). Furthermore, when the recognition site of the limited degradation protease is present between the target protein and the chaperonin subunit, the chaperonin-protein complex of this embodiment having a ring-shaped structure may be treated with the limited degradation protease. The recognition site is not digested.

  The target protein in the chaperonin-protein complex of the present invention is not particularly limited, but there are proteins that tend to form inclusion bodies, proteins that are highly hydrophobic, proteins that are toxic to host cells, proteins that are susceptible to protease attack, etc. Suitable as target protein. In particular, examples of proteins that are highly hydrophobic and easily insolubilized include transmembrane proteins and membrane-bound proteins. More specifically, examples include G protein-coupled receptors (7-transmembrane receptors) such as human endothelin A receptor, ion channel receptors, tyrosine kinase receptors, and the like.

  In the chaperonin-protein production method of the present invention, it is particularly preferable to express the fusion protein in a host cell. Examples of the host cell to be used include bacteria, yeast, plant cells, animal cells and the like, and bacteria that have a high growth rate and can be easily cultured are preferred. Furthermore, Escherichia coli is particularly preferred because it has a rich so-called host-vector system. In order to express the fusion protein in the host cell, it is necessary to introduce a fusion gene encoding the fusion protein into the host cell. In this case, for example, an appropriate expression vector that can replicate in the host cell is used. A recombinant can be prepared by integrating the fusion gene and introducing the expression vector into a host. For example, examples of expression vectors that can be replicated in E. coli include ColEI, pBR, and pACYC plasmid vectors. Further, multiple copies of the expression vector are advantageous for mass production.

In the method for producing a chaperonin-protein of the present invention, there are two methods for synthesizing a single chaperonin subunit. One is a method in which a gene encoding a single chaperonin subunit is incorporated into an appropriate vector capable of replicating in the host cell, the expression vector is introduced into the host cell, and the single chaperonin subunit is expressed. When using this method, a method of further incorporating a single chaperonin subunit gene under the control of a promoter controlling the expression of the fusion gene on the same expression vector in which the fusion gene is incorporated, on the same expression vector There are a method of incorporating under the control of different promoters, and a method of incorporating into a vector that can coexist in the same host cell, unlike an expression vector into which a fusion gene has been incorporated. In the case of Escherichia coli, the above-described ColE1, pBR, and pACYC plasmid vectors can coexist in E. coli.

  Another method for synthesizing a single chaperonin subunit is to express the chaperonin subunit inherently possessed by the host cell from the genomic DNA of the host cell. According to this method, a complicated operation of cloning a single chaperonin subunit, incorporating it into an expression vector, and introducing it into a host cell is unnecessary. Furthermore, since the host cell only needs to hold the expression vector having the fusion gene, the burden on the host cell is reduced, and the recombinant can be cultured more stably and easily. In the case of this method, the expression of chaperonin from genomic DNA can be induced by heat shock by utilizing the fact that chaperonin is a heat shock protein. Examples of the method for applying heat shock include temporarily raising the culture temperature during cultivation of the recombinant, and when the recombinant is Escherichia coli, the culture temperature may be temporarily raised to about 42 ° C. .

  Although all the examples of the above-described chaperonin-protein production methods use host cells, the present invention is not limited to them, and a cell-free translation system can also be used. In this case, a fusion gene encoding a fusion protein and a gene encoding a single chaperonin subunit may be added to the cell-free extract to express these genes. The cell-free extract may further contain RNA polymerase, various nucleotide triphosphates, various amino acids, various tRNAs, ribosomal fractions, various phosphorylases and the like.

  As a method for purifying the chaperonin-protein complex of the present invention, various methods generally used for protein purification can be applied. For example, a purified chaperonin-protein complex can be obtained by combining techniques such as ammonium sulfate fractionation, various types of chromatography, and ultrafiltration. Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography and the like. In the case of a chaperonin-protein complex composed of a thermostable chaperonin subunit, other contaminating proteins can be precipitated and removed by subjecting the sample to heat treatment before purification, which facilitates purification. The gene of the thermostable chaperonin subunit can be obtained from, for example, genomic DNA of thermostable bacteria.

The method for producing a target protein of the present invention is to cut out and isolate a target protein from a fusion protein in which one chaperonin subunit or a chaperonin subunit conjugate and a target protein are linked via a peptide bond. It includes three main steps. The first step is a step in which a fusion protein and a single chaperonin subunit are associated non-covalently to form a chaperonin-protein complex. That is, the first step is mainly intended to correctly fold the target protein to obtain a normal protein having a correct three-dimensional structure. As a method for forming the chaperonin-protein complex, the same configuration as the above-described method for producing the chaperonin-protein complex of the present invention can be applied. That is, the fusion protein is particularly preferably expressed in the host cell, and Escherichia coli is preferably used as the host. The single chaperonin subunit may be expressed by introducing an expression vector incorporating the gene into the host cell, or the host cell's inherent chaperonin subunit may be expressed as genomic DNA of the host cell. May be expressed from In this step, by forming a chaperonin-protein complex, even if the target protein is toxic to the host cell, its toxic expression can be suppressed, and the target protein is susceptible to protease attack. Even so, the target protein is protected without attack. On the other hand, a fusion protein and a single chaperonin subunit can be expressed in a cell-free translation system to form a chaperonin-protein complex. The chaperonin-protein complex thus obtained is subjected to the second step.

  The second step is a step of releasing the fusion protein by dissociating the chaperonin-protein complex formed in the first step into a fusion protein and a single chaperonin subunit. That is, the second step is intended to release the fusion protein from the chaperonin-protein complex so that the target protein can be excised from the fusion protein. In order to dissociate the chaperonin-protein complex into a fusion protein and a single chaperonin subunit, non-covalent association forces between the fusion protein and a single chaperonin subunit, ie, hydrogen bonding, hydrophobic interaction , Treatment to weaken ionic bond, van der Waals force, etc. Specifically, the non-covalent association force is weakened by the addition of a denaturing agent, addition of a surfactant, addition of a chelating agent, removal of adenosine triphosphate, heating, etc., and the chaperonin-protein complex Can be dissociated into a fusion protein and a single chaperonin subunit. Examples of denaturing agents include urea and guanidine hydrochloride. These treatments are preferably performed under mild conditions so that the target protein is not irreversibly denatured. For example, urea is preferably used at a concentration of 3M or less.

  The third step is a step of cutting out and isolating the target protein from the fusion protein released in the second step. That is, the third step is aimed at recovering the target protein. Examples of a method for cutting out the target protein include a method using a limited degradation protease. That is, first, a fusion protein in which a chaperonin subunit and a target protein are linked via a recognition site of a limited degradation protease is used. In this step, the limited degradation protease is allowed to act on the released fusion protein, the recognition site is digested, and the target protein can be cut out. Examples of methods other than the treatment with limited-degradation protease include limited degradation with cyanogen bromide and the like, and methods using self-cleaving peptides such as intein.

  In the case of using the above-mentioned limited degradation protease, even if the limited degradation protease is allowed to act on the chaperonin-protein complex immediately after the first step, the protease recognition site is not digested, and the target protein Cannot be cut out. That is, it is necessary to release the fusion protein from the chaperonin-protein complex in the second step and cause the protease to act on the fusion protein. This is considered to be because the protease recognition site is not exposed to the outside in the chaperonin-protein complex described above and is not affected by the protease.

  The method for producing a target protein of the present invention can be applied to the production of all target proteins, and is particularly suitable for proteins with high hydrophobicity, proteins that are toxic to host cells, and proteins that are susceptible to protease attack. .

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Isolation of GroEL subunit gene Genomic DNA was extracted and purified from Escherichia coli HMS174 (DE3) strain (Novagen). Next, PCR was performed using the purified genomic DNA as a template and the oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 as a primer pair, and a DNA fragment containing the GroEL subunit gene having the nucleotide sequence shown in SEQ ID NO: 3 was obtained. Amplified.

2. Isolation of human endothelin A receptor gene PCR was performed using a human placenta cDNA library (Takara Bio Inc.) as a template and oligonucleotides having the base sequences shown in SEQ ID NOs: 4 and 5 as primer pairs, and the base shown in SEQ ID NO: 6 _A DNA fragment containing the endothelin A receptor (ET _A R) gene having the sequence was amplified. This amplified DNA fragment was provided with a BglII site at the 5 ′ end and a sequence encoding a FLAG tag (FLAG Tag, SEQ ID NO: 7) at the 5 ′ end and an XhoI site derived from the primer.

3. Construction of each vector for expressing fusion protein of GroEL subunit N-fold ligation (N = 1-6) and human endothelin A receptor Plasmid pTrc99A (Amersham Biosciences) was digested with restriction enzymes NcoI and XbaI. After dephosphorylating the ends with bacterial alkaline phosphatase (BAP), the oligonucleotide shown in SEQ ID NO: 8 was inserted to construct plasmid pTrc99ATC. In addition, the NcoI site, the XbaI site, the base sequence encoding the thrombin recognition site, the BglII site, the XhoI site, and the stop codon were introduced into the plasmid pTrc99ATC by the oligonucleotide shown in SEQ ID NO: 8. Next, pTrc99ATC was digested with the restriction enzyme XbaI, the end was dephosphorylated with BAP, and then the DNA fragment containing the GroEL subunit gene amplified in 1 above was inserted to construct plasmid pTrcG ₁ TC. The plasmid pTrcG ₁ TC contains one GroEL subunit gene, and has a restriction enzyme site into which a gene encoding the target protein can be inserted downstream thereof.

Next, the plasmid pTrcG ₁ TC was digested with XbaI, the end was dephosphorylated with BAP, and then the DNA fragment containing the GroEL subunit gene amplified in 1 above was inserted to construct plasmid pTrcG ₂ TC. The plasmid pTrcG ₂ TC has a restriction enzyme site into which a gene encoding a target protein can be inserted downstream of a gene in which two GroEL subunit genes encoding a double-ligated GroEL subunit are connected in tandem. Have. Furthermore, various plasmids of pTrcG ₃ TC, pTrcG ₄ TC, pTrcG ₅ TC, and pTrcG ₆ TC were constructed in the same procedure. Hereinafter, these six types of plasmids are collectively referred to as “pTrcG _N TC (N = 1 to 6)”.

Furthermore, six types of plasmids pTrcG _N TC (N = 1 to 6) were respectively converted into restriction enzymes Bg.
was digested with lII and XhoI, it was dephosphorylated end at BAP, by inserting the amplified DNA fragment containing the ET _A R obtained above 2, of the six expression vectors pTrcG _N TCF-ET _A R (N = 1-6) was constructed. Figure 6 shows the structure of _{pTrcG N TCF-ET A R (} N = 1~6). In the figure, groEL _n encodes a GroEL subunit N-linked, N
Represents a gene in which a plurality of GroEL subunit genes are linked in tandem, “thrombin recognition site” represents a base sequence encoding a thrombin recognition site, ET _A R represents an ET _A R gene, and a FLAG tag represents a FLAG tag gene Represents. ATG represents a start codon, and TAATAG represents a base sequence including a stop codon. _{That, pTrcG N TCF-ET A R} (N = 1~6) is downstream of the trc promoter, in turn, initiation codon, gene encoding the GroEL subunits N times connecting body (N = 1 to 6), of thrombin It has a base sequence encoding a recognition site, an ET _A R gene, a FLAG tag gene, and a stop codon. That is, p
According to _{TrcG N TCF-ET A R (} N = 1~6), capable of expressing the fusion protein of GroEL subunits N times connecting body (N = 1 to 6) and ET _A R.

4). GroEL subunits N times connecting body (N = 1 to 6) and human endothelin A 6 kinds of expression vectors obtained in Preparation 3 above of each recombinant that expresses a fusion protein with receptors pTrcG _N TCF-ET _A R a (N = 1 to 6) each E. coli BLR (DE3) strain was introduced into (Novagen), six recombinant E. coli _{BLR (DE3) / pTrcG N TCF} -ET a R (N = 1~6) Obtained.

5. Expression of the fusion protein six recombinant E. coli _{BLR (DE3) / pTrcG N TCF} -ET A R a (N = 1~6), respectively, 2 × YT medium (16g / L Bacto tryptone containing 100 [mu] g / mL carbenicillin 10 g / L yeast extract, 5 g / L NaCl) was cultured at 25 ° C. for 24 hours. After completion of the culture, each bacterial cell was collected by centrifugation (30000 g, 30 minutes, 4 ° C.). Each collected bacterial cell was washed with PBS, suspended in a suspension buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl ₂ , 1 mM EDTA), and disrupted by ultrasonication. Each cell disruption solution was centrifuged (100,000 g, 1 hour, 4 ° C.), and the supernatant was collected to obtain each cell extract. These bacterial cell extracts were each subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) (SDS-PAGE / CBB). As a control, the same operation was performed for the plasmid pTrc99ATC into which the gene encoding the fusion protein was not inserted. The results are shown in each lane 1 of FIG. FIG. 7 shows the results of SDS-PAGE / CBB in the case of control, N = 1, 2, 3, 4, 5, 6 in order from the left. Lane 6 is a molecular weight marker. That is, the position of the black arrow corresponding to the molecular weight of each fusion protein G _N -ET _A R (N = 1 to 6) (G ₁ -ET _A R: 109 kDa, G ₂ -ET _A R: 166 kDa, G ₃ -ET _A R: 223 kDa, G ₄ -ET _A R: 280 kDa, G ₅ -E
T _A R: 337 kDa, G ₆ -ET _A R: 394 kDa), a band not seen in the control was confirmed.

Furthermore, each bacterial cell extract obtained was subjected to immunoprecipitation using an anti-FLAG antibody-immobilized gel (Sigma). The suspension buffer was used for washing, and the elution fraction was eluted using the suspension buffer containing 100 μg / mL FLAG peptide (SEQ ID NO: 7). The results of subjecting the non-adsorbed fraction, the washed fraction, and the adsorbed fraction to SDS-PAGE / CBB are shown in each lane 2, each lane 3, and each lane 4 in FIG. That is, a band corresponding to the fusion protein G _N -ET _A R (N = 1 to 6) was detected only in the adsorbed fraction (lane 3). In addition to G _N -ET _A R is the adsorbed fraction, a band was also detected at the same time corresponding to a single GroEL subunits having no FLAG tag (white arrow). From these facts, it was confirmed that the fusion protein and a single GroEL subunit were associated non-covalently.

Further, by the action of thrombin adsorbed fraction above was confirmed whether ET _A R is cut from _{G N -ET A R (N =} 1~6). As reaction conditions, 2 units of thrombin (Amersham Biosciences) was added to 10 mg (converted to BSA) of G _N -ET _A R (N = 1 to 6) and treated at 25 ° C. for 6 hours. The results are shown in each lane 5 of FIG. That is, a band corresponding to the ET _A R in either case (42 kDa) was not _{detected, G N -ET A R (N} = 1
ET _A R from 6) was cut out. Than, be _{G N -ET A R (N =} 1~6) and the sole GroEL subunits forms a complex, which ET _A R is stored in the interior of the ring-shaped structure confirmed more It was done.

The same SDS-PAGE as described above was performed again, and Western blotting was performed. A PVDF membrane (Immobilon-P, Millipore) was used for blotting. As a primary antibody, a monoclonal antibody against FLAG peptide (ANTI-FLAG M2 Monoclonal Antibody, Sigma) was used. As a secondary antibody, a biotin-labeled universal secondary antibody kit (Universal Quick Kit, Vector Laboratories) was used. For detection, Konica Immunostain HRP-100 (Seikagaku Corporation) was used. As a result, it is band appeared at a position corresponding to a molecular weight of the fusion protein _{G N -ET A R (N =} 1~6) , are both _{G N -ET A R (N =} 1~6) confirmed.

6). Production of GroEL-human endothelin A receptor complex Among 6 types of prepared recombinant E. coli, N = 3 E. coli BLR (DE3) / pTrcG ₃ TCF-ET _A 2 × 100 μg / mL carbenicillin Incubated with 1 L of YT medium. After completion of the culture, the cell disruption supernatant was collected in the same manner as in 5 above. The obtained supernatant was subjected to anti-FLAG antibody-immobilized gel (ANTI-FLAG M2-Agarose) according to the following procedure.
(from volume mouse, Sigma)) (column volume: 8 mL, column inner diameter: 1 cm). First, the column was equilibrated with an equilibration buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA), and the resulting supernatant was applied. After washing the column with the equilibration buffer, stepwise elution was performed with the equilibration buffer containing 100 μg / mL FLAG peptide (Sigma). The flow rate was 2 mL / min and the column temperature was about 25 ° C. (room temperature). Each fraction was analyzed by SDS-PAGE / CBB to identify fractions containing the fusion protein G ₃ -ET _A R and collected them.

Next, ammonium sulfate was added to the collected fraction to a final concentration of 10% (w / v) and subjected to hydrophobic interaction chromatography using a HiTrap Butyl FF 5 mL column (Amersham Bioscience). For elution, buffer A (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl ₂ , 1 mM EDTA, 1
mM DTT, 10% (w / v) ammonium sulfate) and buffer B (50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl ₂ , 1 mM EDTA, 1 mM
DTT), and eluted with a linear gradient of buffer B from 0 to 100%. The flow rate was 3 mL / min and the column temperature was 4 ° C. Each fraction was analyzed by SDS-PAGE / CBB to identify fractions containing the fusion protein G ₃ -ET _A R and collected them.

Next, after the collected fraction was concentrated by ultrafiltration (Centriprep YM-50, molecular weight cut off 50000, Millipore), a TSK G4000SW _XL column (diameter 7.5 mm, length 30 cm, Tosoh Corporation) was used. Was subjected to gel filtration chromatography. As the developing solution, a buffer composed of 25 mM HEPES-KOH (pH 7.0), 300 mM NaCl, 40 mM KCl, 5 mM MgCl2, 1 mM EDTA, and 1 mM DTT was used. The flow rate was 0.5 mL / min. As a control, the same operation was performed for N = 7. The results of gel filtration chromatography are shown in FIGS. 8 (a) and 8 (c). In the figure, the vertical axis represents absorbance (A ₂₈₀ ) at a wavelength of 280 nm, and the horizontal axis represents elution time (minutes). That is, in the case of N = 3 (FIG. 8 (a)), as in the case of N = 7 (FIG. 8 (c)), the elution volume which is the elution position of the ring-shaped structure composed of 14 GroEL subunits. A peak was detected around 8.6 mL (0.16 CV) (Weissman, JS et al. Cell 84, 481-490 (1996)). Furthermore, SDS-PAGE / CBB was performed for each fraction at the peak position of gel filtration chromatography. The results are shown in FIGS. 8 (b) and 8 (d). That is, in the case of N = 3 (FIG. 8B), a band (black arrow) corresponding to G ₃ -ET _A R (223 kDa) is detected, and in the case of N = 7 as a control (FIG. 8D). ), _A band (black arrow) corresponding to G ₇ -ET _A R (451 kDa) was detected. From the above, it was confirmed that the fusion protein G ₃ -ET _A R forms a double ring structure complex consisting of a total of 14 GroEL subunits with a single GroEL subunit.

The fraction containing the peak in FIG. 8 (a) was negatively stained with 0.2% uranyl acetate, and the morphology was observed with a transmission electron microscope. The photograph is shown in FIG. As shown in FIG. 5, a ring-shaped structure peculiar to chaperonin or chaperonin-protein complex was observed. This indicates that the fusion protein G ₃ -ET _A R forms a chaperonin-protein complex (GroEL-human endothelin A receptor complex) with a GroEL subunit derived from the recombinant genomic DNA, and It was found that the composite formed a ring-shaped structure.

1. Production of human endothelin A receptor In the same manner as in Example 1, N = 3 E. coli BLR (DE3) / pTrcG ₃ TCF-
ET _A R was cultured to obtain a bacterial cell extract. Furthermore, in the same manner as in Example 1, the bacterial cell extract was subjected to various chromatography to obtain a gel filtration fraction containing GroEL-human endothelin A receptor complex. Urea was added to this fraction to a final concentration of 3 M and treated at room temperature for 30 minutes to dissociate GroEL-human endothelin A receptor complex into fusion protein G ₃ -ET _A R and GroEL subunit, The fusion protein G ₃ -ET _A R was released. Next, this reaction solution was dialyzed several times against PBS using a dialysis membrane (molecular weight cut off 10,000) to remove urea. Then, thrombin was added to a G ₃ -ET _A R to 10 units of 10 mg (BSA equivalent), was treated 0-6h at 25 ° C., a part of the reaction mixture by SDS-PAGE / CBB analyzed. As a control, the same operation was performed for N = 7. The results are shown in FIGS. 9 (a) and 9 (c). In the figure, lane 1 is 0 minutes, lane 2 is 15 minutes, lane 3 is 30 minutes, lane 4 is 1 hour, lane 5 is 1.5 hours, lane 6 is 2 hours, lane 7 is 2.5 hours, lane 8 is a sample after 3 hours, lane 9 is 4 hours, lane 10 is 5 hours, and lane 11 is a sample after each reaction time of 6 hours. That is, when N = 3, a band was observed in the vicinity of 42 kDa corresponding to ET _A R over a reaction time of 1 hour (lane 4) to 6 hours (lane 11), and G ₃ -ET _A R to ET _A R Could be cut out (FIG. 8A). In particular, when the reaction time was 6 hours, the band corresponding to G ₃ -ET _A R disappeared, G ₃ -ET _A R was completely digested by thrombin, and ET _A R was completely excised. This result was the same as in the case of control N = 7 (FIG. 9C). Thus, a fusion protein G ₃ -ET _A R is the thrombin recognition site is cleaved by thrombin, resulting could be isolated by cutting out the ET _A R from the fusion protein G ₃ -ET _A R.

When urea treatment was not performed, no band corresponding to ET _A R was observed in SDS-PAGE in any case, and the thrombin recognition site was not cleaved by thrombin (FIG. 9 (b), FIG. 9). (D)). This indicates that the GroEL-human endothelin A receptor complex is not digested by thrombin, but the released fusion protein is digested by thrombin. The absence of thrombin digestion of the GroEL-human endothelin A receptor complex indicates that the thrombin recognition site is not exposed to the outside, which indicates that the GroEL-human endothelin A receptor complex is in a ring shape. This is one proof of having a structure.

It is a schematic diagram which shows the structural example of the chaperonin-protein complex of this invention, (a)-(f) shows the example of N = 1-6 of a chaperonin subunit N times coupling body, respectively. It is a schematic diagram which shows another structural example of the chaperonin-protein complex of this invention. It is a schematic diagram which shows another structural example of the chaperonin-protein complex of this invention, (a) shows the example which the target protein has couple | bonded with the both ends of the chaperonin subunit coupling body, (b) is the target protein. Shows examples in which are bound to separate chaperonin subunit conjugates. It is a schematic diagram which shows another example of a structure of the chaperonin-protein complex of this invention, (a) is a kind with which the chaperonin subunit which comprises the chaperonin subunit coupling body, and a single chaperonin subunit differ. An example is shown, and (b) shows an example in which different chaperonin subunits are mixed in both a chaperonin subunit linked body and a single chaperonin subunit. It is a photograph which shows the result of having observed the chaperonin-protein complex of this invention with the electron microscope. It is a schematic diagram showing a structure of a plasmid _{pTrcG N TCF-ET A R (} N = 1~6). Extract of recombinant Escherichia coli expressing a fusion protein of N-fold GroEL subunit N (N = 1 to 6) and ET _A R, anti-FLAG antibody non-adsorbed fraction, same washed fraction, same adsorbed fraction It is a photograph which shows the result which used the minute and the thrombin processing sample for SDS-PAGE. It is a photograph which shows the result when the chromatogram when the sample containing a GroEL-human endothelin A receptor complex was used for gel filtration, and each fraction of gel filtration was used for SDS-PAGE, (a) is N = 3. (B) shows the result of subjecting the fraction corresponding to (a) to SDS-PAGE, and (c) and (d) show the results of a control experiment when N = 7. . It is a photograph showing the result of subjecting the GroEL-human endothelin A receptor complex to thrombin digestion after urea treatment or without urea treatment, and subjected to SDS-PAGE. (A) Example of urea treatment with N = 3 (B) shows an example in which N = 3 and no urea treatment was performed, and (c) and (d) show the results of a control experiment when N = 7. It is a schematic diagram which shows the three-dimensional structure of Escherichia coli GroEL.

Claims (27)

  In a chaperonin-protein complex formed by assembling a plurality of chaperonin subunits and a target protein, a part of the plurality of chaperonin subunits and a target protein form a fusion protein, and the fusion protein includes one A chaperonin subunit or a chaperonin subunit conjugate and a target protein are linked via a peptide bond, and the remaining chaperonin subunits are linked to the target protein and other chaperonin subunits via a peptide bond. A chaperonin-protein complex, wherein the chaperonin subunit is a non-covalent chaperonin subunit, and the fusion protein and the single chaperonin subunit are associated non-covalently.   The chaperonin-protein complex according to claim 1, wherein the number of the target proteins is one.   The chaperonin-protein complex according to claim 1 or 2, wherein the fusion protein is a chaperonin subunit or chaperonin subunit conjugate and a target protein linked via a linker peptide. body.   The chaperonin-protein complex according to claim 3, wherein the linker peptide includes a recognition site for a limited-degradation protease.   5. The chaperonin-protein complex according to claim 4, wherein the limited degradation protease is thrombin.   The chaperonin-protein complex according to claim 4 or 5, wherein the recognition site is not cleaved by the action of the limited degradation protease.   The chaperonin-protein complex according to any one of claims 1 to 6, wherein the plurality of chaperonin subunits are subunits of a group 1 type chaperonin.   The chaperonin-protein complex according to claim 7, wherein all of the subunits of the group 1 type chaperonin are derived from the same organism.   8. The chaperonin-protein complex according to claim 7, wherein all subunits of the group 1 type chaperonin are derived from E. coli.   The chaperonin-protein complex according to any one of claims 1 to 9, wherein the total number of the plurality of chaperonin subunits is an optimal number determined by the origin of the chaperonin subunits.   The chaperonin-protein complex according to claim 10, wherein the optimal number is seven.   The chaperonin- of any one of claims 1 to 11, wherein the plurality of chaperonin subunits form a ring-shaped structure having a cavity therein, and the target protein is stored in the cavity. Protein complex. The chaperonin-protein complex according to claim 12, wherein a ring-shaped structure formed by another chaperonin subunit is associated non-covalently with the ring-shaped structure.   The chaperonin-protein complex according to any one of claims 1 to 13, wherein the target protein is a transmembrane receptor.   The chaperonin-protein complex according to claim 14, wherein the transmembrane receptor is a G protein-coupled receptor.   The chaperonin-protein complex according to claim 15, wherein the G protein-coupled receptor is a human endothelin A receptor.   In a method for producing a chaperonin-protein complex in which a plurality of chaperonin subunits and a target protein are assembled, a gene encoding one chaperonin subunit or a chaperonin subunit conjugate and a gene encoding the target protein are linked. The synthesized fusion gene is transcribed and translated to synthesize a fusion protein in which one chaperonin subunit or chaperonin subunit conjugate and the target protein are linked via peptide bonds, and the fusion protein and a single chaperonin subunit are synthesized. A method for producing a chaperonin-protein complex, wherein the unit is associated non-covalently.   18. A recombinant having the fusion gene is cultured, the fusion gene is transcribed and translated in the recombinant, and the chaperonin-protein complex is obtained from the recombinant culture. A method for producing the chaperonin-protein complex described in 1.   The method for producing a chaperonin-protein complex according to claim 18, wherein the fusion gene is incorporated into an expression vector.   The chaperonin according to claim 18 or 19, wherein a single chaperonin subunit is synthesized by transcription and translation of a gene encoding a chaperonin subunit present on the genomic DNA of the recombinant. -A method for producing a protein complex.   The chaperonin-protein complex according to claim 18 or 19, wherein a single chaperonin subunit is synthesized by transcription and translation of a gene encoding a chaperonin subunit incorporated into an expression vector. Production method.   The method for producing a chaperonin-protein complex according to any one of claims 18 to 21, wherein the recombinant is Escherichia coli. In a method for producing a target protein, the target protein is cut out and isolated from a fusion protein in which one chaperonin subunit or chaperonin subunit conjugate and the target protein are linked via a peptide bond.
(1) non-covalently associating the fusion protein with a single chaperonin subunit to form a chaperonin-protein complex;
(2) dissociating the chaperonin-protein complex formed in step (1) into a fusion protein and a single chaperonin subunit, and releasing the fusion protein;
(3) A step of cutting out and isolating the target protein from the fusion protein released in step (2),
A method for producing a target protein, comprising: In the step (1), a recombinant having a fusion gene encoding the fusion protein is cultured, and the fusion gene is transcribed and translated in the recombinant to form a chaperonin-protein complex. The method for producing a target protein according to claim 23.   The method for producing a target protein according to claim 24, wherein the recombinant is Escherichia coli.   The method for producing a target protein according to any one of claims 23 to 25, wherein in step (2), the chaperonin-protein complex is dissociated by the action of a denaturing agent.   The fusion protein is obtained by binding one chaperonin subunit or a chaperonin subunit conjugate and a target protein through a recognition site of a limited-degraded protease, and in the step (3), The method for producing a target protein according to any one of claims 23 to 26, wherein the recognition site is cleaved and the target protein is cut out and isolated from the fusion protein.