JP2013199457A - Chaperonin complex and method of producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chaperonin complex capable of encapsulating metal nano particles with excellent efficiency.SOLUTION: A chaperonin complex comprises: a chaperonin variant; and metal nano particles. The chaperonin variant comprises a plurality of GroEL subunits containing at least one GroEL subunit variant selected from the group comprising: a first GroEL subunit variant that consists of a specific amino acid sequence; and a second GroEL subunit variant that consists of an amino acid sequence in which one or more amino acid residues other than an alanine residue lying at a specific position are substituted, deleted, or added, among the amino acid sequences, and that has molecular chaperone activity. The metal nano particles are surrounded with the plurality of GroEL subunits.

Description

  The present invention relates to a chaperonin complex and a method for producing the same.

Chaperonins are a class of so-called molecular chaperones that help the correct folding of substrate proteins. The chaperonin family is a protein having a molecular weight of 50 kDa to 60 kDa, and has a common feature of taking a ring-shaped complex structure and assisting the folding of the substrate protein in an ATP-dependent manner. Among the chaperonins, GroEL is a chaperonin possessed by Escherichia coli, and it has been clarified that it assists the folding of the substrate protein depending on ATP and GroES.
The wild-type chaperonin GroEL has a 14-mer structure in total, in which a 7-mer of GroEL subunits constitutes one ring and this ring is further overlapped two back to back. That is, the wild-type chaperonin GroEL is surrounded by seven GroEL subunits and has two cavities that can contain a substrate protein. One GroEL subunit is composed of an equator domain containing an ATP binding site, a vertex domain containing a binding site between a substrate protein and GroES, and an intermediate domain connecting both domains.

In the folding of the substrate protein, when the substrate protein first binds to the “entrance” of the ring composed of the chaperonin GroEL subunit, and the seven ATPs bind to the chaperonin GroEL subunit constituting the ring, the structural change of the chaperonin GroEL Occurs and GroES, which is a cofactor, can bind to GroEL. Then, when GroES binds to GroEL, the substrate protein is dropped into the ring cavity, forming a chaperonin complex. In the chaperonin complex, the folding of the substrate protein dropped into the ring cavity proceeds. Then, when ATP in the ring is hydrolyzed, GroES dissociates, and at the same time, the folded substrate protein in the ring also dissociates. That is, the time for hydrolysis of ATP is a timer for the reaction cycle of chaperonin GroEL.
The hydrolysis time of ATP is about 8 seconds for wild type GroEL. On the other hand, among the amino acid sequence of wild-type GroEL, a GroEL mutant in which the aspartic acid residue at No. 398 is substituted with an alanine residue (hereinafter also referred to as “GroEL (D398A)”), etc. has an ATP hydrolysis activity. It is known that the half-life of the complex is 30 minutes or more, as compared to the wild type (see, for example, Non-Patent Document 1 and Patent Document 1).

  In addition, it is known that wild-type chaperonin forms a complex containing semiconductor nanoparticles, and the semiconductor nanoparticles can be released in the presence of ATP (see Non-Patent Document 2).

JP 2008-294487 A

Cell, Vol. 97, p325-338, 1999. Nature, Vol. 423, p628-632, 2003.

Since the chaperonin complex encapsulating semiconductor nanoparticles described in Non-Patent Document 2 is formed by spontaneous interaction of chaperonin and semiconductor nanoparticles, it is possible to obtain a chaperonin complex encapsulating semiconductor nanoparticles with high efficiency. There were cases where it was difficult.
An object of the present invention is to provide a chaperonin complex capable of encapsulating metal nanoparticles with excellent efficiency, a method for producing the chaperonin complex, and a substrate having the chaperonin complex.

Specific means for solving the above problems are as follows.
That is, the first aspect of the present invention is the first GroEL subunit variant consisting of the amino acid sequence of SEQ ID NO: 1, and 1 or 2 other than the alanine residues at positions 52 and 398 in the amino acid sequence of SEQ ID NO: 1. Containing at least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant having a molecular chaperone activity, comprising an amino acid sequence in which the above amino acid residues are substituted, deleted, or added. A chaperonin complex having a chaperonin variant composed of a plurality of GroEL subunits and metal nanoparticles surrounded by the plurality of GroEL subunits. The chaperonin complex preferably further includes at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof, and is preferably at least one selected from the group consisting of ADP, ATP, and derivatives thereof. More preferably, it further comprises a GroES subunit in addition to the nucleotide.

  The second aspect of the present invention is the first GroEL subunit variant consisting of the amino acid sequence of SEQ ID NO: 1, and one or more than the alanine residues at positions 52 and 398 in the amino acid sequence of SEQ ID NO: 1 A plurality of amino acid residues comprising at least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant having a molecular chaperone activity. A chaperonin complex comprising a chaperonin mutant consisting of the GroEL subunits and metal nanoparticles, and surrounding the metal nanoparticles with the plurality of GroEL subunits. The chaperonin mutant and the metal nanoparticle are preferably contacted in the presence of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof and a GroES subunit.

  3rd aspect of this invention is a board | substrate provided with a support body and the said chaperonin complex arrange | positioned on the said support body. The chaperonin complex preferably includes a GroEL subunit group that surrounds the metal nanoparticles and a GroEL subunit group that does not surround the metal nanoparticles.

  ADVANTAGE OF THE INVENTION According to this invention, the board | substrate which has a chaperonin complex which can enclose a metal nanoparticle with the outstanding efficiency, its manufacturing method, and this chaperonin complex can be provided.

It is a figure which shows an example of the transmission electron microscope image of the chaperonin complex concerning the Example of this invention. It is a figure which shows an example of the transmission electron microscope image of the chaperonin complex concerning the Example of this invention. It is a figure which shows an example of the transmission electron microscope image of the bullet-shaped chaperonin complex concerning the Example of this invention.

<Chaperonin complex>
The chaperonin complex of the present invention includes a first GroEL subunit variant consisting of the amino acid sequence of SEQ ID NO: 1 and one or more than one of the amino acid sequences of SEQ ID NO: 1 except for the 52nd and 398th alanine residues. A plurality of amino acid residues comprising at least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant having an amino acid sequence substituted, deleted, or added and having molecular chaperone activity It has a chaperonin variant composed of GroEL subunits and metal nanoparticles surrounded by the plurality of GroEL subunits. That is, the chaperonin complex is a metal nanoparticle-containing chaperonin complex in which metal nanoparticles are encapsulated in a cavity surrounded by a plurality of GroEL subunits.
A chaperonin mutant comprising a GroEL subunit mutant having a specific structure can generate a chaperonin complex in which metal nanoparticles are encapsulated with excellent efficiency. Moreover, it is excellent in the uniformity of the particle diameter of the metal nanoparticle included. Furthermore, the chaperonin complex can release the encapsulated metal nanoparticles after a predetermined time.

  This can be considered as follows, for example. Nature, Vol. The complex described in 423, p628-632 (2003) is formed by spontaneous interaction between chaperonin molecules and CdS nanoparticles in the absence of ATP or the like, and thus it is difficult to say that the production efficiency is sufficient. On the other hand, since the chaperonin complex of the present invention has a chaperonin mutant including a GroEL subunit mutant in which the hydrolysis rate of ATP is suppressed, it can be formed in the presence of ATP and the GroES subunit. is there. Thereby, it can be considered that the metal nanoparticles are efficiently encapsulated in the chaperonin mutant. When the chaperonin complex further includes a GroES subunit, it can be considered that the interaction between the chaperonin mutant and the metal nanoparticle is enhanced.

  The chaperonin mutant takes in metal nanoparticles in a dispersed state to form a chaperonin complex. That is, since the chaperonin mutant does not form a chaperonin complex with the aggregated metal nanoparticles, it can be considered that the particle size of the encapsulated metal nanoparticles is uniform.

  In the present specification, a GroEL subunit multimer comprising a plurality of GroEL subunits and having a cavity surrounded by the GroEL subunit is referred to as “chaperonin GroEL”, and at least one GroEL subunit variant is defined as a GroEL subunit. The chaperonin GroEL containing is called “chaperonin mutant”, and the complex of chaperonin mutant and metal nanoparticles is called “chaperonin complex”. A numerical range expressed using “to” means a range including numerical values described before and after “to” as a lower limit value and an upper limit value. Further, the amount of each component in the composition means the total amount of the plurality of substances present in the composition unless there is a specific notice when there are a plurality of substances corresponding to each component in the composition. Further, the term “process” is not limited to an independent process, and is included in this term if the intended action of the process is achieved even when it cannot be clearly distinguished from other processes.

(Chaperonin mutant)
The GroEL subunit mutant contained in the chaperonin mutant is a first GroEL subunit mutant having the amino acid sequence of SEQ ID NO: 1, and other than the 52nd and 398th alanine residues in the amino acid sequence of SEQ ID NO: 1. Is at least one selected from the group consisting of a second GroEL subunit variant consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, or added.
The second GroEL subunit mutant is not particularly limited as long as the ATP hydrolysis activity is lower than that of the wild-type GroEL subunit. In the second GroEL subunit mutant, the number of amino acid residue substitutions, deletions, or additions at positions other than the 52nd and 398th alanine residues in the amino acid sequence of SEQ ID NO: 1 It is preferably 15 or less, more preferably 10 or less, and still more preferably 5 or less.

Specific examples of the substitution of the amino acid residue include the following.
In general, in order to maintain the function of a protein, the amino acid residue to be substituted is preferably an amino acid residue having properties similar to the amino acid residue before substitution. Such substitution of amino acid residues is called conservative substitution. For example, Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are all classified as nonpolar amino acid residues, and thus have similar properties to each other. Examples of the non-charged amino acid residue include Gly, Ser, Thr, Cys, Tyr, Asn, and Gln. Moreover, Asp and Glu are mentioned as an acidic amino acid residue. Examples of basic amino acid residues include Lys, Arg, and His. Substitution of amino acid residues within each of these groups is preferably tolerated.

  The substitution of the amino acid residue may add a function to the second GroEL subunit mutant. Specific examples of the substitution that adds a new function include, for example, a mutant obtained by mutating the aspartic acid residue at position 490 of wild-type GroEL to a cysteine residue (Nat. Biotechnol., 2001 Sep; 19 (9): 861-5.). Such a mutation enables the chaperonin mutant to be immobilized on a glass substrate or the like. Moreover, the mutant (Biochem. Biophys. Res. Commun. 2000 Jan 27; 267 (3): 842-9.) Which mutated the asparagine residue of No. 265 to an alanine residue can also be mentioned. Such a mutation allows the metal nanoparticles to be more efficiently encapsulated in the chaperonin mutant.

The second GroEL subunit mutant may have the same function as the first GroEL subunit mutant, or may have a function added thereto. Specific examples thereof include a mutant (Cell, 2006 Jun 2; 125 (5): 903-14.) In which the C-terminal repetitive sequence in the GroEL subunit is deleted and added. Such a mutation makes it possible to change the volume of the cavity of the chaperonin mutant.
Further, the second GroEL subunit variant may be one in which one or more amino acid residues are further added depending on the use. Examples of such amino acid residues that can be added include amino acid residues that can constitute a signal peptide, a tag sequence, and the like.

  The second GroEL mutant subunit may have a mutation other than the mutation mentioned in the specific example. Examples of such mutation include Cell. 2002 Dec 27; 111 (7): 1027-39. 7 consisting of a single ring described in Cell. 1995 Nov 17; 83 (4): 577-87. Examples include mutations that make it possible to form a monomer.

The GroEL subunit mutant can be produced, for example, by expressing a DNA comprising a base sequence encoding the GroEL subunit mutant by a commonly used method. Specifically, it is produced by infecting a host cell selected according to the recombinant vector with a recombinant vector containing a DNA consisting of a nucleotide sequence encoding a GroEL subunit mutant, and culturing the host cell. be able to.
For the details of the production method of the GroEL subunit mutant, for example, the production method described in JP-A-2008-294487 can be referred to.

  The chaperonin mutant is a first GroEL subunit mutant consisting of the amino acid sequence of SEQ ID NO: 1, and one or more amino acid residues other than the 52 and 398 alanine residues in the amino acid sequence of SEQ ID NO: 1. It comprises at least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant having a molecular chaperone activity, consisting of an amino acid sequence in which a group is substituted, deleted, or added. That is, at least one of the plurality of (preferably 7, more preferably 14) GroEL subunits constituting the chaperonin mutant is the above GroEL subunit mutant.

The chaperonin mutant has a significantly reduced ATP hydrolysis activity as compared with a conventionally known chaperonin mutant (for example, GroEL (D398A)). Therefore, the formed chaperonin complex can encapsulate metal nanoparticles stably for a predetermined time.
The chaperonin complex can encapsulate metal nanoparticles in the presence of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof and a GroES subunit. The efficiency is high, and chaperonin complexes can be generated with excellent efficiency.
Further, when the chaperonin mutant is a 14-mer of the GroEL subunit, the metal nanoparticles can be encapsulated simultaneously in the cavities of the two rings constituting the chaperonin mutant, so that the metal nanoparticles are more efficiently chaperonin. It can be encapsulated in a complex.

The number of GroEL subunits constituting the chaperonin mutant is not particularly limited as long as it can encapsulate metal nanoparticles. The chaperonin mutant is preferably a 7-mer of the GroEL subunit, more preferably a 14-mer. The total number of GroEL subunit variants in the GroEL subunit group constituting the chaperonin variant may be at least 1. When the chaperonin mutant is a 14-mer of GroEL subunit, the total number of GroEL subunit mutants is preferably 7 or more, more preferably 14, from the viewpoint of ATP hydrolysis activity.
The chaperonin mutant is formed from a group of GroEL subunits including a GroEL subunit mutant under normal conditions, for example, ATP-dependent (Nature, 1990 Nov 22; 348 (6299): 339-42). The

(Metal nanoparticles)
The chaperonin complex includes at least one metal nanoparticle. The metal nanoparticles are not particularly limited as long as they are nanoparticles containing a metal element and can be included in the chaperonin mutant, and can be appropriately selected according to the purpose. The metal nanoparticles are substantially composed of metal nanoparticles composed of a single metal element, an alloy including two or more metal elements, or a metal nanoparticle composed of a metal compound, and a metal compound including a metal element and a non-metal element. Any of metal nanoparticles etc. may be sufficient. The metal nanoparticles may be either magnetic particles or diamagnetic particles, and may be any of conductor particles and semiconductor particles.
Here, “substantially” means not excluding the inclusion of other elements inevitably mixed. Specifically, the content of other elements is 1% by mass or less.

Examples of the metal element constituting the metal nanoparticles include at least one metal element selected from the group consisting of the third period, the fourth period, the fifth period, and the sixth period of the Long Periodic Table (IUPAC 1991). be able to. Among them, at least one metal selected from Groups 2 to 14 is preferable, and Group 2, Group 4, Group 5, Group 6, Group 7, Group 8, Group 9, Group 10, Group 10 is preferable. More preferably, at least one metal element selected from Group 11, Group 12, Group 13, and Group 14 is used.
Specific examples of the metal element include copper, silver, gold, platinum, palladium, nickel, tin, cobalt, rhodium, iridium, iron, ruthenium, osmium, manganese, molybdenum, tungsten, niobium, tantalum, titanium, bismuth, Examples include antimony, lead, silicon, germanium, cadmium, indium, and chromium.

Examples of the alloy and metal compound include FePt, CdS, CdSe, and SiO ₂ .

The average particle diameter of the metal nanoparticles is not particularly limited as long as it can be included in the chaperonin mutant. For example, the thickness is preferably 1 nm to 10 nm, and more preferably 2 nm to 5 nm. The average particle diameter of the metal nanoparticles is obtained as an arithmetic average value of the particle diameters of the metal nanoparticles measured as a circle-equivalent particle diameter for 350 particles with a transmission electron microscope (TEM).
The metal nanoparticles may be primary particles composed of one metal nanoparticle or secondary particles composed of a plurality of metal nanoparticles.

When the chaperonin mutant is a 14-mer of GroEL subunit, the chaperonin mutant has two cavities that can encapsulate metal nanoparticles. In the chaperonin complex, the metal nanoparticles may be contained in at least one of the two cavities. By encapsulating the metal nanoparticles in both of the two cavities, a chaperonin complex having a high content of metal nanoparticles can be obtained. Moreover, it can be set as the chaperonin complex which is excellent in the adhesiveness to a board | substrate by including a metal nanoparticle only in one cavity.
In addition, when the chaperonin mutant can encapsulate two metal nanoparticles, each metal nanoparticle may be the same or different.

  The number of metal nanoparticles encapsulated in one cavity of the chaperonin mutant is appropriately selected according to the particle diameter of the metal nanoparticles. From the viewpoint of making the particle diameter of the metal nanoparticles encapsulated uniform, the number of metal nanoparticles encapsulated in one cavity is preferably one.

  It is preferable that the chaperonin complex further includes at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof. When the chaperonin complex further includes at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof, a structural change occurs in the chaperonin complex, and the metal nanoparticles are more efficiently converted into the chaperonin mutant. Can be included.

  Examples of the ADP or ATP derivative include ADPBeFx, AMPBeFx, ATPγS, an ATP substitute compound described later, and the like. For example, when the chaperonin complex contains ADPBeFx as a nucleotide, football-type and bullet-type chaperonin complexes described later can be further stabilized.

The content of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof in the chaperonin complex is, when the chaperonin mutant is a 14-mer of GroEL subunit, per chaperonin mutant molecule. It is preferably 7 or 14 molecules, and when the nucleotide is ADP or a derivative thereof, there are 7 molecules per chaperonin variant, and when the nucleotide is ATP or a derivative thereof, a chaperonin variant is 1 molecule. More preferably, it is 14 molecules.
When the nucleotide is a derivative of ADP or ATP, the number of nucleotides contained in the chaperonin complex is preferably selected according to the type of derivative.

  The chaperonin complex preferably further has a GroES subunit in addition to at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof. By further including the GroES subunit, the chaperonin mutant can encapsulate the metal nanoparticles more efficiently, and the generation efficiency of the chaperonin complex is further improved. The content of the GroES subunit in the chaperonin complex is preferably 7 or 14 molecules per molecule of the chaperonin mutant when the chaperonin mutant is a 14-mer of the GroEL subunit, and the nucleotide is ADP or In the case of the derivative, there are 7 GroES subunits per molecule of chaperonin mutant, and in the case where the nucleotide is ATP or a derivative thereof, there are 14 GroES subunits per molecule of chaperonin mutant. More preferred.

  The GroES subunit acts as a cofactor of chaperonin GroEL, and can encapsulate metal nanoparticles, which are inclusions, in the cavity of chaperonin GroEL. In the present invention, the GroES subunit may be a wild-type GroES subunit or a GroES subunit mutant. In addition, a fluorescent label or the like may be added to the GroES subunit by a conventional method. As the fluorescent label or the like, a commonly used fluorescent label or the like can be used without particular limitation.

  The GroES subunit may be derived from Escherichia coli or manufactured by a commonly used method. The GroES subunit can be produced, for example, by expressing DNA consisting of a base sequence encoding the GroES subunit by a commonly used method. Specifically, it can be produced by infecting a host cell selected according to the recombinant vector with a recombinant vector containing a DNA consisting of a base sequence encoding a GroES subunit and culturing the host cell. it can.

  Since the chaperonin complex of the present invention includes metal nanoparticles, it can be used as a metal nanoparticle dispersion excellent in dispersion stability in a dispersion medium. Such a chaperonin complex can be used for applications such as cosmetics.

<Method for producing chaperonin complex>
The method for producing the chaperonin complex of the present invention comprises the first GroEL subunit variant consisting of the amino acid sequence of SEQ ID NO: 1, and 1 or aside from the 52nd and 398th alanine residues in the amino acid sequence of SEQ ID NO: 1. At least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant having a molecular chaperone activity, comprising an amino acid sequence in which two or more amino acid residues are substituted, deleted, or added. An encapsulating step of contacting a metal nanoparticle with a chaperonin mutant composed of a plurality of GroEL subunits and surrounding the metal nanoparticle with the plurality of GroEL subunits. The method for producing the chaperonin complex may further include other steps as necessary.

  The method for bringing the chaperonin mutant into contact with the metal nanoparticles is not particularly limited. For example, a method of mixing a chaperonin mutant and metal nanoparticles in a suitable buffer can be mentioned. As a method of mixing the chaperonin mutant and the metal nanoparticles, it can be appropriately selected from commonly used stirring methods. For example, a method of mixing by pipetting in a microtube, a method of mixing using a rotator, a method of mixing using vortex, and the like can be mentioned. As temperature which makes a chaperonin variant and a metal nanoparticle contact, it can be 4-60 degreeC, for example, and it is more preferable that it is 25-37 degreeC. The contact time is preferably 1 to 60 minutes, for example, and more preferably 1 to 2 minutes.

As said buffer solution, it can select from the buffer solution used normally, and can use it suitably. For example, HEPES, PBS, Tris, etc. can be mentioned.
The pH of the buffer solution is preferably 5 to 9, and more preferably 7 to 8. For adjusting the pH, for example, an inorganic base such as KOH or NaOH or an inorganic acid such as HCl can be used.
It is preferable that the buffer solution further contains an inorganic salt. Examples of the inorganic salt include KCl, MgCl ₂ , Na ₂ SO _{4 and the} like. Among these, from the viewpoint of the production efficiency of the chaperonin complex, it is preferable to contain KCl and MgCl ₂ .

The concentration of the buffer solution is preferably 1 mM to 100 mM, and more preferably 20 mM to 40 mM, from the viewpoint of the production efficiency of the chaperonin complex.
When the buffer solution contains KCl as an inorganic salt, the concentration of KCl is preferably 1 mM to 200 mM, and more preferably 50 mM to 100 mM. Also when the buffer containing MgCl ₂ as the inorganic salt, it is preferable that the concentration of MgCl ₂ is 1 mm to 10 mm, and more preferably from 4 mm to 6 mm.

  The mixing ratio of the chaperonin mutant and the metal nanoparticles in the encapsulation step is not particularly limited. For example, from the viewpoint of the generation efficiency of the chaperonin complex, 1 g to 10 g of metal nanoparticles are preferably mixed per 1 μmol of GroEL subunit constituting the chaperonin mutant, and more preferably 2 g to 8 g are mixed.

  The encapsulation step is preferably performed in the presence of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof, and at least one selected from the group consisting of ADP, ATP, and derivatives thereof And in the presence of a GroES subunit, and at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof, a GroES subunit, and a metal ion (preferably More preferably, it is carried out in the presence of magnesium ions). As a result, a chaperonin complex in which metal nanoparticles are encapsulated can be produced with excellent production efficiency.

When the chaperonin mutant has two cavities and the encapsulation process is performed in the presence of ATP, a football-type chaperonin complex in which metal nanoparticles are encapsulated in both of the two cavities of the chaperonin mutant is preferential. Can get to.
On the other hand, when the chaperonin mutant has two cavities and the encapsulation step is performed in the presence of ADP, a bullet-shaped chaperonin complex in which metal nanoparticles are encapsulated only in one of the two cavities of the chaperonin mutant. Can be preferentially obtained.

  Further, the encapsulation step is performed in the presence of the chaperonin mutant, the first metal nanoparticles, and the ADP, and the first chaperonin in which the first metal nanoparticles are encapsulated in one of the two cavities of the chaperonin mutant. After obtaining the complex, the first chaperonin complex and the second metal nanoparticle are brought into contact with each other in the presence of ATP, so that the first metal nanoparticle and the second metal nanoparticle are 1 A hybrid chaperonin complex encapsulated in two chaperonin mutants can also be obtained.

  In the encapsulation step, an ATP substitute compound may be used instead of ATP. The ATP surrogate compound is not particularly limited as long as it is a compound capable of binding to the ATP binding site of the GroEL subunit mutant and causing the structural change of the chaperonin GroEL mutant. For example, an adduct of ADP and beryllium fluoride (J. Biol. Chem., 279, 45737-45743 (2004).), An adduct of ADP and aluminum fluoride or gallium fluoride (J. Mol. Biol., 2003) May 23; 329 (1): 121-34.).

When the inclusion step is performed in the presence of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof, the GroEL subunit is selected from the group consisting of ADP, ATP, and derivatives thereof. It is preferable to use at least one kind of nucleotide in a molar ratio of 10 ³ to 10 ⁶ , and more preferable to use a molar ratio of 10 ³ to 10 ⁴ .

  When the encapsulation step is performed in the presence of the GroES subunit, the GroES subunit is preferably used in a molar ratio of 1 to 10 and more preferably in a molar ratio of 1 to 3 with respect to the GroEL subunit.

  When the encapsulation step is performed in the presence of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof, and a GroES subunit, a chaperonin mutant, a metal nanoparticle, ADP, ATP And the order of mixing the at least one nucleotide selected from the group consisting of these derivatives and the GroES subunit is not particularly limited, and these may be mixed simultaneously or sequentially. Above all, from the viewpoint of the generation efficiency of the chaperonin complex, at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof is mixed in the mixture of the chaperonin mutant, the metal nanoparticle, and the GroES subunit. It is preferable that it is a method to do.

The method for producing a chaperonin complex may further include a purification step, a modification step, and the like of the chaperonin complex in addition to the encapsulation step.
Examples of the purification step include a method of separation by chromatography, a method of ultrafiltration, and the like.

<Board>
The board | substrate of this invention is equipped with a support body and the said chaperonin complex arrange | positioned on the said support body. The substrate may further include other components as necessary.
In the said board | substrate, it arrange | positions on a support body in the state in which aggregation of the chaperonin complex which includes a metal nanoparticle was suppressed. That is, the metal nanoparticles can be arranged on the support in a state in which the aggregation is suppressed.

  The support is not particularly limited, and can be appropriately selected from commonly used supports according to the purpose. Examples of the support include a glass substrate, a resin substrate such as a phenol resin, a metal substrate such as alumina, and carbon.

The arrangement mode of the chaperonin complex on the support is not particularly limited. For example, when the chaperonin complex is the football type, it is preferable that the long axis direction of the football type is arranged substantially parallel to the surface of the support. When the chaperonin complex is a bullet type, it is preferable that the chaperonin complex is disposed so that a cavity that does not contain metal nanoparticles is in contact with the support.
By using a bullet-type chaperonin complex as a chaperonin complex in this way, the chaperonin complex encapsulating metal nanoparticles on the support is arranged in a planar shape with the cavity enclosing the metal nanoparticles facing upward can do. This can be attributed to, for example, the hydrophobic interaction between the chaperonin complex and the support.

The substrate is formed by, for example, applying a dispersion containing the chaperonin complex and a dispersion medium on a support, removing at least a part of the dispersion medium, and adsorbing the chaperonin complex on the support. Can be manufactured.
The dispersion medium is not particularly limited as long as the chaperonin complex can be dispersed, and can be appropriately selected from commonly used dispersion media. Examples of the dispersion medium include water, a buffer solution, and ethanol. The method and conditions for removing the dispersion medium are not particularly limited, and can be appropriately selected according to the use of the dispersion medium and the substrate used.

  The substrate of the present invention can be suitably used for applications such as sensors because the chaperonin complex encapsulating metal nanoparticles is disposed on the support.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, “%” is based on mass. When a commercially available kit was used in this example, the operation was performed according to the instruction manual for the kit.

[Preparation Example 1]
A GroEL subunit consisting of the amino acid sequence of SEQ ID NO: 1 (hereinafter also referred to as “GroEL subunit mutant”) was prepared according to the method described in Japanese Patent Application Laid-Open No. 2008-294487.

[Preparation Example 2]
FePt nanoparticles were prepared as metal nanoparticles that were magnetic as follows.
(1) Synthesis of unmodified FePt nanoparticles In a three-necked flask, 0.39 g (1.0 mmol / l) of acetylacetone platinum (II), 0.35 g (1.0 mmol / l) of acetylacetone iron (II), 100 ml of tetraethylene glycol Were mixed and replaced with gas in an Ar gas atmosphere for 1 hour. The mixture was refluxed at 300 ° C. for 1 hour in an Ar atmosphere. Then, it cooled to room temperature in Ar atmosphere, and prepared the tetraethylene glycol dispersion liquid of the unmodified FePt nanoparticle.
An equal amount of ethanol was mixed with the obtained tetraethylene glycol dispersion containing the unmodified FePt nanoparticles, centrifuged at 4000 rpm × 20 minutes, and then the supernatant was removed to obtain a precipitate. Ethanol washing, in which 50 ml of ethanol was added to the resulting precipitate and centrifuged at 4000 rpm × 20 minutes to remove the supernatant, was repeated until the centrifuged supernatant was no longer colored.
The finally obtained precipitate was dispersed in ultrapure water, HKM buffer, or ethanol to obtain an unmodified FePt nanoparticle dispersion. The resulting unmodified FePt nanoparticle dispersion was stored at 4 ° C.
The composition of the HKM buffer is 20 mM HEPES, 100 mM KCl, 5 mM.
MgCl ₂ and adjusted to pH 7.5 with KOH.

(2) Synthesis of pyruvate-modified FePt nanoparticles In a three-necked flask, 0.39 g (1.0 mmol / l) of acetylacetone platinum (II), 0.35 g (1.0 mmol / l) of acetylacetone iron (II), tetraethylene glycol 100 ml was mixed and replaced with gas in an Ar gas atmosphere for 1 hour. After refluxing at 300 ° C. for 1 hour in an Ar atmosphere, the temperature was lowered to 150 ° C. After adding 20 ml of a mixed solution of pyruvic acid and tetraethylene glycol (1: 1 (v / v), prepared by stirring for 12 hours), the mixture was refluxed again for 2 hours. Then, it cooled to room temperature in Ar atmosphere, and prepared the pyruvic acid modification FePt nanoparticle.
The obtained tetraethylene glycol dispersion of pyruvate-modified FePt nanoparticles was washed in the same manner as described above to obtain a pyruvate-modified FePt nanoparticle dispersion.

(3) Synthesis of oleic acid-modified FePt particles In a three-necked flask, 0.39 g (1.0 mmol / l) of acetylacetone platinum (II), 0.35 g (1.0 mmol / l) of acetylacetone iron (II), 100 ml of tetraethylene glycol Were mixed and replaced with gas in an Ar gas atmosphere for 1 hour. After refluxing at 300 ° C. for 1 hour in an Ar atmosphere, the temperature was lowered to 200 ° C. After adding 10 ml of oleic acid, the mixture was refluxed again for 30 minutes. Then, it cooled to room temperature in Ar atmosphere, and prepared the oleic acid modification FePt nanoparticle.
The resulting tetraethylene glycol dispersion of oleic acid-modified FePt nanoparticles was washed in the same manner as described above to obtain an oleic acid-modified FePt nanoparticle dispersion.

Each of the FePt nanoparticle dispersions obtained above was placed on a collodion support membrane grid and dried for 24 hours to obtain an observation sample. The obtained sample for observation was observed with a transmission electron microscope JEM 2000EX (manufactured by JEOL Ltd.) at an acceleration voltage of 100 kV to obtain a particle electron microscope image. The obtained particle electron microscopic image was taken in at 600 dpi, and subjected to particle analysis (Analyze Particles) using image processing software Image J (Ver. 1.46), and an average particle size was determined for each FePt nanoparticle dispersion.
As a result, the unmodified FePt nanoparticles had an average particle size of 4.88 nm, the pyruvate-modified FePt nanoparticles had an average particle size of 3.99 nm, and the oleic acid-modified FePt nanoparticles had an average particle size of 5.05 nm.

[Example 1]
Using a non-modified FePt nanoparticle HKM buffer dispersion (concentration 100 mg / ml), a chaperonin complex was prepared as follows.
Each material was added to the microtube so that the final concentrations were as follows, and mixed with a micropipette at room temperature for 1 minute to prepare a sample solution.
・ GroEL subunit mutant 0.1 μM
・ GroES subunit 0.2μM
・ ATP 1 mM
・ Unmodified FePt nanoparticles 2.5mg / ml

  6.0 μl of the sample solution obtained above was brought into contact with the hydrophilized collodion support membrane for 1 minute. After rinsing once with 6.0 μl of ultrapure water, 6.0 μl of 2% uranium acetate was added and stained for 1 minute. After drying overnight, it was observed at an acceleration voltage of 80 kV using a transmission electron microscope HT-7500 (manufactured by Hitachi High-Tech). An example of the obtained observation image is shown in FIG.

  From FIG. 1, a football-type chaperonin complex in which two unmodified FePt nanoparticles are encapsulated in a chaperonin mutant, and a bullet-shaped chaperonin complex in which one unmodified FePt nanoparticle is encapsulated in a chaperonin mutant Can be observed. That is, the football-type chaperonin complex is shown, for example, in the enlarged view at the lower right of FIG. The bullet-shaped chaperonin complex is shown, for example, in the enlarged view in the upper left of FIG.

[Example 2]
In Example 1, instead of unmodified FePt nanoparticles, an HKM buffer dispersion of pyruvate modified FePt nanoparticles (concentration 100 mg / ml) was used, and the final concentration of pyruvate modified FePt nanoparticles was 3.5 mg / ml. A sample solution was prepared in the same manner as in Example 1 except that the above was obtained.
When observed with a transmission electron microscope (acceleration voltage 120 kV) in the same manner as in Example 1, a chaperonin complex in which pyruvate-modified FePt nanoparticles are encapsulated in a chaperonin mutant is obtained as in Example 1. Observed.
In the obtained chaperonin complex, the magnetism of the pyruvate-modified FePt nanoparticles was maintained.

[Example 3]
In Example 1, a sample solution was prepared in the same manner as in Example 1, except that an HKM buffer dispersion (concentration: 100 mg / ml) of oleic acid-modified FePt nanoparticles was used instead of unmodified FePt nanoparticles. .
When observed with a transmission electron microscope in the same manner as in Example 1, a chaperonin complex in which oleic acid-modified FePt nanoparticles were encapsulated in the chaperonin mutant was observed as in Example 1.

[Example 4]
20.0 μl of HKM buffer dispersion of pyruvate modified FePt nanoparticles (concentration 20.0 mg / ml), 0.8 μl of HKM buffer containing 5.0 μM GroEL subunit variant, 10.0 μM GroES subunit The HKM buffer solution 0.8 μl was mixed by pipetting in a microtube for 1 minute, and then stirred at room temperature for 40 minutes with a rotator (RT-5 (Titec), 4 r / min). Thereafter, 17.99 μl of HKM buffer and 0.41 μl of 96.6 mM ATP solution were added, and the sample solution was further stirred at room temperature with a rotator (RT-5 (Titec), 4 r / min) for 20 minutes. . The final concentration in 40.0 μl of this sample solution was as follows.
・ GroEL subunit mutant 0.1 μM
・ GroES subunit 0.2μM
・ ATP 1 mM
・ Pyruvate-modified FePt nanoparticles 10mg / ml

6.0 μl of the sample solution obtained above was brought into contact with the hydrophilized collodion support membrane for 1 minute. After rinsing once with 6.0 μl of ultrapure water, 6.0 μl of 1.0% phosphotungstic acid (pH 4.0) was added and stained for 1 minute. After drying for 16 hours or more, observation was performed using a transmission electron microscope JEM2000EX (manufactured by JEOL Ltd.) at an acceleration voltage of 100 kV. An example of the obtained observation image is shown in FIG.
From the obtained observation images, the number of chaperonin complexes encapsulating metal nanoparticles in 350 chaperonin complexes and the number of chaperonin complexes in which the metal nanoparticles are not encapsulated are counted, respectively. When the encapsulation rate was calculated, it was 95.4%.

[Example 5]
12.5 μl of HEPES buffer (pH 7.5) dispersion of pyruvate-modified FePt nanoparticles (concentration: 40.0 mg / ml), 2.0 μl of HKM buffer containing 5.0 μM GroEL subunit variant, 10.0 μM The HKM buffer containing 2.0 μl of GroES subunit and 33.5 μl of HKM buffer were mixed by pipetting for 1 minute in a microtube. Thereafter, 48.96 μl of HKM buffer and 1.04 μl of 96.6 mM ATP solution were added, and the sample solution was further prepared by pipetting at room temperature for 20 seconds. The final concentration in 100.0 μl of this sample solution was as follows.
・ GroEL subunit mutant 0.1 μM
・ GroES subunit 0.2μM
・ ATP 1 mM
・ Pyruvate-modified FePt nanoparticles 5mg / ml
When the encapsulation rate of metal nanoparticles in 609 chaperonin complexes was calculated in the same manner as described above, it was 89.5%.

[Example 6]
Unmodified FePt nanoparticle dispersion (concentration: 40.0 mg / ml, 40 mM HEPES-KOH, pH 7.5) 6.25 μl, HKM buffer containing 5.0 μM GroEL subunit variant 10.0 μl, 10.0 μM 10.0 μl of HKM buffer containing GroES subunit was mixed by pipetting for 1 minute in a microtube. Next, 29.48 μl of HKM buffer and 0.52 μl of 96.6 mM ATP solution were added, and further pipetting was performed at room temperature for 20 seconds to prepare a sample solution. The final concentration in 50.0 μl of this sample solution was as follows.
・ GroEL subunit mutant 1.0μM
・ GroES subunit 2.0μM
・ ATP 1.0 mM
・ Unmodified FePt nanoparticles 5mg / ml

6.0 μl of the sample solution obtained above was brought into contact with the hydrophilized collodion support membrane for 40 seconds. After rinsing once with 6.0 μl of ultrapure water, 6.0 μl of 1.0% phosphotungstic acid (pH 4.0) was added and stained for 1 minute. After drying for 16 hours or more, observation was performed using a transmission electron microscope HT-7500 (manufactured by Hitachi High-Tech) at an acceleration voltage of 80 kV.
As a result, the chaperonin complex formed in the presence of ATP as shown in the enlarged view at the lower right of FIG. 1 has a football-type shape, and the football-type long axis is on the support surface with the surface of the support. It was arrange | positioned so that it might become substantially parallel.

[Example 7]
Unmodified FePt nanoparticle dispersion (concentration: 40.0 mg / ml, 40 mM HEPES-KOH, pH 7.5) 6.25 μl, HKM buffer containing 5.0 μM GroEL subunit variant 10.0 μl, 10.0 μM 10.0 μl of HKM buffer containing GroES subunit was mixed by pipetting for 1 minute in a microtube. Next, 29.48 μl of HKM buffer and 0.51 μl of 99.1 mM ADP solution were added, and the sample solution was further pipetted at room temperature for 20 seconds. The final concentration in 50.0 μl of this sample solution was as follows.
・ GroEL subunit mutant 1.0μM
・ GroES subunit 2.0μM
・ ADP 1.0 mM
・ Unmodified FePt nanoparticles 5mg / ml

6.0 μl of the sample solution obtained above was brought into contact with the hydrophilized collodion support membrane for 40 seconds. After rinsing once with 6.0 μl of ultrapure water, 6.0 μl of 1.0% phosphotungstic acid (pH 4.0) was added and stained for 1 minute. After drying for 16 hours or more, observation was performed using a transmission electron microscope HT-7700 (manufactured by Hitachi High-Tech) at an acceleration voltage of 80 kV. An example of the obtained observation image is shown in FIG.
From FIG. 3, the chaperonin complex formed in the presence of ADP has a bullet shape, and is arranged on the support so that the long axis of the bullet is substantially perpendicular to the surface of the support. I understand.

[Example 8]
Quantum dot CdS 460 (Lumidot ^™ Lumidot ^™ , manufactured by Sigma-Aldrich) having a fluorescence wavelength of 460 nm is commercially available after being dispersed in toluene because it is modified with carboxylic acid. The dispersion medium was changed as follows.
100.0 μl of CdS 460 toluene dispersion (CdS concentration 5.0 mg / ml) was placed in a microtube, and 1.0 ml of ethanol was added thereto and mixed gently. After centrifugation at 14500 rpm × 10 minutes, the supernatant was removed. 500.0 μl of methanol was added to the obtained yellow precipitate, and the mixture was uniformly dispersed by irradiating with ultrasonic waves for about 1 minute to obtain a CdS nanoparticle dispersion having a concentration of 1.0 mg / ml.

40.0 μl of HKM buffer containing 2.5 μM GroEL subunit variant, 40.0 μl of HKM buffer containing 5.0 μM GroES subunit, and 8.96 μl of HKM buffer were mixed in a microtube. Next, 10.0 μl of CdS nanoparticle dispersion (concentration 1.0 mg / ml, methanol) was added and pipetted for 1 minute, then 1.04 μl of 96.6 mM ATP solution was added, and further piped for 20 seconds at room temperature. A sample solution was prepared by petting. The final concentration in 100.0 μl of this sample solution was as follows.
・ GroEL subunit mutant 1.0μM
・ GroES subunit 2.0μM
・ ATP 1.0 mM
・ CdS nanoparticles 0.1mg / ml

  6.0 μl of the sample solution obtained above was brought into contact with the hydrophilized collodion support membrane for 40 seconds. After rinsing once with 6.0 μl of ultrapure water, 6.0 μl of 1.0% phosphotungstic acid (pH 4.0) was added and stained for 1 minute. After being dried for 16 hours or more, and observed with a transmission electron microscope JEM2000EX (manufactured by JEOL Ltd.) at an acceleration voltage of 80 kV, as in Example 1, a chaperonin complex in which CdS nanoparticles were encapsulated in a chaperonin mutant was obtained. Observed.

  From the above results, it can be seen that the chaperonin complex of the present invention is excellent in complex formation efficiency. It can also be seen that by forming a chaperonin complex in the presence of ATP, a football-type chaperonin complex in which metal nanoparticles are encapsulated in both of the two cavities of the chaperonin mutant is obtained. On the other hand, it can be seen that by forming a chaperonin complex in the presence of ADP, a bullet-shaped chaperonin complex in which metal nanoparticles are encapsulated in one of the two cavities of the chaperonin mutant is obtained.

Claims (7)

In the first GroEL subunit variant consisting of the amino acid sequence of SEQ ID NO: 1, and in the amino acid sequence of SEQ ID NO: 1, one or more amino acid residues other than the 52nd and 398th alanine residues are substituted or deleted Or a chaperonin variant comprising a plurality of GroEL subunits comprising at least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant comprising an added amino acid sequence and having molecular chaperone activity When,
Metal nanoparticles surrounded by the plurality of GroEL subunits;
A chaperonin complex having:   The chaperonin complex according to claim 1, further comprising at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof.   The chaperonin complex according to claim 2, further comprising a GroES subunit. In the first GroEL subunit variant consisting of the amino acid sequence of SEQ ID NO: 1, and in the amino acid sequence of SEQ ID NO: 1, one or more amino acid residues other than the 52nd and 398th alanine residues are substituted or deleted Or a chaperonin variant comprising a plurality of GroEL subunits comprising at least one GroEL subunit variant selected from the group consisting of a second GroEL subunit variant comprising an added amino acid sequence and having molecular chaperone activity When,
Metal nanoparticles,
And contacting the metal nanoparticles with the plurality of GroEL subunits to produce a chaperonin complex.   The contact between the chaperonin mutant and the metal nanoparticle is performed in the presence of at least one nucleotide selected from the group consisting of ADP, ATP, and derivatives thereof and a GroES subunit. Of producing a chaperonin complex.   A board | substrate provided with a support body and the chaperonin complex of any one of Claims 1-3 arrange | positioned on the said support body.   The substrate according to claim 6, wherein the chaperonin complex includes a GroEL subunit group that surrounds the metal nanoparticles and a GroEL subunit group that does not surround the metal nanoparticles.