JP2002209588A - Method for stabilizing protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for stabilizing a useful protein with a chaperonin without occasionally supplying a nucleotide, and for effectively utilizing the characteristics of the protein. SOLUTION: This method for stabilizing the protein is characterized by continuously holding the natural type protein molecule in the hollow of the group 2 type chaperonin molecule.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for preventing a natural protein from being denatured by environmental stress such as heat and maintaining the protein in a stable state.

[0002]

2. Description of the Related Art Proteins are polypeptides in which a plurality of amino acids are linked by peptide bonds. In order for a protein to exhibit its properties, a specific tertiary structure (steric structure) formed by intramolecular or intermolecular interaction is important. In general, when an environmental stress such as heat is applied to a natural protein, its tertiary structure changes, and its characteristics often disappear irreversibly (denatured protein). Therefore, how to keep the native protein stable against such environmental changes has always been cited as an issue in handling proteins.

[0003] As a solution to the above-mentioned problems, intensive studies have been made so far, and various improvement plans have been proposed.
In recent years, interest has been increasing in molecular chaperones as factors involved in the formation and structural changes of proteins. Chaperonins, one of molecular chaperones, are a group of heat shock proteins, which are accumulated in cells when cells are exposed to various environmental stresses such as temperature changes. These are widely present in eubacteria, archaebacteria, and eukaryotes, and have been shown to be involved in the formation of three-dimensional structures of proteins regardless of the type of protein. Gr as a chaperonin produced especially by eubacteria
oEL is well known. For example, GroEL of Escherichia coli has a characteristic structure composed of a total of 14 subunits, in which a donut-shaped structure in which seven subunits are connected in a ring is overlapped in two stages. GroEL is known to trap a denatured protein in the cavity of a donut structure and efficiently fold it into a protein with the correct tertiary structure as a result of hydrolysis of nucleotides such as ATP and binding of cofactor GroES. ing. Some attempts have been made to apply this chaperonin to protein stabilization. For example, JP-A-7-67641
Japanese Patent Application Laid-Open Publication No. HEI 9-264, proposes a "method of stabilizing an enzyme in a solution by adding a nucleotide such as a chaperonin protein and ATP to an enzyme-containing solution." Also, JP-A-7-48
Japanese Patent Publication No. 398 discloses a method for regenerating an inactive protein that has been chemically denatured, an inactive protein accumulated in a transformant used in genetic engineering, and the like into an active protein.・ Thermofilas ( Th
ermus thermophilus ) using a purified chaperonin ”.

[0004] Each of these uses the action of forming a three-dimensional structure of a protein possessed by a chaperonin. When the three-dimensional structure is deformed by heat or a denaturing agent, the polypeptide chain of the protein is unwound into its original three-dimensional structure (folding) ) The purpose is achieved by the action.

However, chaperonins are generally AT
Hydrolysis of nucleotides such as P (adenosine-5 'triphosphate), CTP (cytidine-5' triphosphate), GTP (guanosine-5 'triphosphate), and UTP (uridine-5' triphosphate) Along with this, the folding reaction proceeds, so that it was necessary to supply expensive nucleotides as needed to stabilize the protein. In addition, these nucleotides are susceptible to self-decomposition due to heat or a pH change accompanying a hydrolysis reaction, and lack economical efficiency. Furthermore, in these methods, the native protein once folded by the chaperonin is released from the chaperonin cavity, and thus has a risk of denaturing again.

[0006]

SUMMARY OF THE INVENTION In view of the above-mentioned problems, the present invention provides a method for stabilizing a useful protein using chaperonin without supplying nucleotides as needed, and for effectively utilizing its properties. With the goal.

[0007]

Generally, after a denatured protein is folded inside a chaperonin in combination with hydrolysis of nucleotides, the denatured protein is released from the cavity as a natural form, and the protein is again exposed to the risk of denaturation. Will be. However, as a result of the inventor's voluntary study, in the folding reaction, when a nucleotide analog was used instead of a nucleotide, the denatured protein was folded at the chaperonin cavity by the nucleotide analog binding to the nucleotide reaction site of the chaperonin, After being converted to the native form, they were found to be continuously trapped inside the chaperonin, thus completing the present invention.
That is, the present invention is a method for stabilizing a protein, which comprises continuously retaining a natural protein molecule inside the cavity of a group 2 type chaperonin molecule.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Chaperonins are classified into two types, group 1 derived from eubacteria and group 2 derived from archaebacteria or eukaryotes (Kim et al. Trends Biochem. Sci. 543-54).
8, 1994). In the present invention, a chaperonin belonging to Group 2 is used. Chaperonins classified into group 1 require the binding of GroES, a 10 kDa subunit hexamer, as a cofactor as a cofactor, as typified by GroEL derived from Escherichia coli. When a protein denatured by heat or the like is folded by chaperonin, the denatured protein is captured in the cavity of the donut structure of chaperonin, but in the case of group 1 type chaperonin, GroES
Closes the chaperonin cavity, for example, when an enzyme is trapped inside chaperonin, the enzyme and the substrate molecule cannot react. On the other hand, the group 2 type chaperonin forms an oligomer with only the 55-60 kDa subunit, as represented by TF55, and exhibits folding activity without a cofactor such as GroES. Therefore, group 2
In the case of type chaperonin, since low-molecular compounds can flow between the chaperonin cavity and the outside, it is possible for the chaperonin cavity to exhibit the intrinsic properties of proteins, such as substance conversion in enzymatic reactions. Become. Normally, when the group 2 type chaperonin captures the denatured protein and the nucleotide coexisting in the reaction solution is hydrolyzed, the quaternary structure of the chaperonin changes, and the unwound protein is released from the molecular cavity. .
Continuous capture of the native protein in the chaperonin cavity is achieved by using homologues of conventional nucleotides such as ATP, GTP, UTP, and CTP. Once bound to the nucleotide reactive site of the chaperonin molecule, the nucleotide homolog does not hydrolyze, so the structural change of the chaperonin is not accompanied, and the folded protein remains in the chaperonin cavity. Specifically, the denatured protein is captured by a chaperonin in a solution, and a folding reaction is performed inside the chaperonin by adding a nucleotide homolog. Nucleotide homologs bind to chaperonins but do not hydrolyze,
The folded native protein remains in the chaperonin cavity as it is and is not released to the outside.

The nucleotide analog used in the present invention is one which binds to the nucleotide reaction site of chaperonin and does not dissociate. As an example, AMP-PNP (5'-
adenylylimido-diphosphate), GMP-PNP (5'-guanylyimi
do-diphosphate), CNP-PNP (5'-cytidylylimido-diphosp
hate), UMP-PNP (5'-uridylylimido-diphosphate) and the like are preferably used. Particularly desirable is, but not limited to, AMP-PNP.

The chaperonin may be derived from eukaryotes, eubacteria and archaebacteria. The chaperonin used in the present invention is a group 2 type chaperonin derived from archaebacteria or eukaryotes. Any chaperonin can be used. Archaeon-derived chaperonins are composed of only a few types of chaperonin monomers, and are therefore convenient when mass-producing chaperonins by genetic recombination using Escherichia coli or the like. In particular, chaperonins from thermophilic or hyperthermophilic archaea are:
Since the chaperonin molecule itself has excellent heat resistance, the protein captured by the chaperonin is also effectively protected from heat stress, and thus can be suitably used. In the present invention, the thermophilic bacterium refers to a microorganism having an optimum growth temperature of 45 to 80 ° C, and the hyperthermophilic bacterium grows at 80 ° C or higher.

The archaea used in the present invention are of the genus Acidianus and Metallosphaerae.
a) the genus, Sutiji Oro bus (Stygiolobus) genus Sulfolobus (Sulfolobus) genus, Sul flow Lactococcus (Sulfurococcu
s) the genus and Frith file gills (Sulfurisphaera) Sul Roman Valles (Sulfolobales) eye, such as the genus, Aeropairamu (A
eropyrum ), Desulfurococcus
Genus, Sutetteria (Stetteria) species, Staphylococcus Thermus
( Staphylothermus ), Thermodiscus
s ), Igneococcus , Thermosphaera and Sulfobococcus
( Sulfophobococcus ) genus, Hyperthermu
s) belonging to the genus, Pyro decrease-tee Umm (Pyrodictium) genus and Pairorobasu (Pyrolobus) species such as IG neo Cocca-less (Igne
ococcules ), Pyrobaculum , Thermoproteus , Thermophilum ( Th
ermofilum) genus and Cardo Lactococcus (Caldococcus) genus Thermo Protea less (Thermoproteales) eye, such as, Akiogurobusu (Archaeoglobus) genus and Ferogurobusu (Ferrog
Archaeoglobales ( Lobus ) and other genus Archaeoglobales
Eyes, Metanosamasu (Methanotherm us) genus, methanolate Agrobacterium (Methanobacterium) genus, methanolate Thermo Enterobacter (M
ethanothermobacter ) and Methanosphaera ( Methanos )
phaera ), such as Methanobacteri
ales) eyes, Methanococcus (Methanococcus) genus, methanolate Thermococcus (Methanothermococcus) genus, methanolate Cardo Lactococcus (Methanocaldococcus) genus and Metanoigunisu (M
ethanoignis ) and other Methanococcales ( Methanococcal
es ) eyes, Methanomicrobiales
Methanosarcinaes ( Methanosarcinaes ), Methanosarcinales eyes, Methanopirales
( Methanopyrales ), Thermococcales such as Pyrococcus and Thermococcus, Thermococcales , Thermoplasma
a ) Thermoplasmales, such as genus and Picrophilus genus;
The chaperonin used in the present invention may be derived from any archaebacteria, but is not limited thereto. Among them, those derived from thermophilic or hyperthermophilic archaebacteria are preferred.

Since the protein that can be stabilized by the present invention must be captured in the cavity of the chaperonin molecule, it is desirable that the protein has a molecular weight of about 60 kDa or less. In order to obtain the chaperonin used in the present invention, a thermophilic archaeon or a hyperthermophilic archaeon is cultured, and the obtained cells are subjected to SDS.
After lysis using a bacterial solubilizing agent, genomic DNA is extracted by a technique such as phenol extraction and ethanol precipitation. This genomic DNA is cleaved with an appropriate restriction enzyme and ligated to an appropriate vector to prepare a genomic DNA library. Vectors include various vectors derived from λ phage, such as phagemid DNA such as λgt10 and λZAP, or pUC18.
Or a plasmid vector such as pBR322. On the other hand, based on the amino acid sequence of a region of high homology between chaperonin genes derived from different species, a DNA corresponding thereto is synthesized and used as a primer for PCR. By performing PCR using the genomic DNA as a template with the primers, a partial fragment of the target gene can be obtained. The partial fragment can be used as a probe for gene screening by labeling it with a radioactive element such as [ ³² P] or a non-radioactive compound such as dicoxigenin.

In order to obtain the entire nucleotide sequence of the target gene, the above-mentioned genomic DNA library may be introduced into a host such as Escherichia coli, and a clone which strongly binds to the above-mentioned labeled probe may be selected. The nucleotide sequence can be determined by a general method such as the Sanger method or the Maxam-Gilbert method. By the above procedure, the entire DNA base sequence encoding the chaperonin including the translation initiation codon to the termination codon can be isolated. By the above operation, the DNA encoding the isolated chaperonin is appropriately pET
The desired chaperonin can be prepared in large quantities by inserting it into an expression vector such as a system and introducing it into a microorganism or cultured cell to express it.

In order to capture the denatured protein with the chaperonin, the denatured protein and the chaperonin oligomer may be mixed in the solution at a mixing ratio of 0.1-10. Preferably, the mixing ratio of the chaperonin oligomer 0.5-2 to the denatured protein 1 is good. Although any buffer solution can be used for the composition of the reaction solution, it is desirable to add potassium ions and magnesium ions. Potassium ion concentration range is 10-100
0 mM and magnesium ion are preferably 10-500 mM.

[0015] After capturing the denatured protein with chaperonin, adding a nucleotide analog causes the protein captured in the chaperonin cavity to fold into a native form, but is released outside the chaperonin as in the case where nucleotides are added. There is no. The addition amount of the nucleotide analog is preferably 0.1 to 10 mM.

The natural protein captured in the group 2 type chaperonin cavity is not isolated from the outside as in the case where the group 2 type chaperonin is captured. It can be used in the same way as ordinary proteins. Since chaperonins obtained from thermophilic archaea and hyperthermophilic archaea are excellent in heat resistance, natural proteins trapped inside also have heat resistance. Therefore, not only can the reaction be performed under a stress environment such as a high temperature, but also it is advantageous during transportation and storage.

[0017]

Hereinafter, the present invention will be described with reference to examples.
The scope of the present invention is not limited to this. Example 1 Preparation of Hyperthermophilic Archaeon Chaperonin A β-chaperonin homo-oligomer derived from the hyperthermophilic archaeon Thermococcus sp. Strain KS-1 was prepared according to the method described in JP-A-10-327869.

0.6 g of the Thermococcus sp.
l TNE buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM
(MEDTA). 10% SDS solution and 1% Triton X-100
Each 1 ml of the solution was added and left at 4 ° C. overnight. Then, the temperature was raised to 50 ° C and the proteinase K solution (20 mg / ml) was added to 0.0
5 ml was added and shaken for 4 hours. After phenol treatment and chloroform treatment, add 0.05 ml of RNase A solution (0.5 mg / ml), and add
It was left at 7 ° C. for 1 hour. Again, 1 ml of 10% SDS solution and 0.05 ml of proteinase K solution (20 mg / ml) were added, and the mixture was left at 50 ° C. for 70 minutes. After phenol and chloroform treatments (solution volume
10 ml), 1 ml of 3M sodium acetate solution (pH 5.2), 25 ml
Was added and left at −20 ° C. for 2 hours. Then, centrifuge with a high-speed centrifuge to precipitate DNA, and add 70% ethanol solution.
Wash the precipitate with 3 ml, dry it with a centrifugal evaporator,
It was dissolved in 0.5 ml of TE buffer. By this operation, about 2 mg of genomic DNA was obtained.

The following DNA primers were synthesized from the amino acid homologous region of archaeal chaperonin. Forward: 5'-CCAAGCTTACNAT (A / T / C) ACNAA (T / C) GA (T / C) G
GNGCNACNAT-3 'Reverse: 5'-ATCTGCAGGA (C / T) TT (C / T) TTNACNC (G / T) NC (G
/ T) NACNGC -3 '

Using the DNA of Thermococcus sp. KS-1 as a template, PCR was performed using a PCR amplification kit (Takara Shuzo). Conditions were as follows: initial denaturation at 94 ° C for 3 minutes, followed by denaturation (94 ° C
Cycle), annealing (55 ° C. for 1 minute), and extension (72 ° C. for 1 minute). The amplified DNA fragment of about 800 bp was ligated to pUC18 and transformed using competent cell E. coli DH5α. Positive clones were selected, and after confirming the presence of the inserted fragment, the cells were inoculated into 2 × YT medium (containing 0.05 mg / ml ampicillin) and cultured with shaking at 37 ° C. overnight. After the culture was collected by centrifugation, a plasmid DNA was prepared. A sequencing reaction was performed using 200 ng of DNA per sample as a template, and the nucleotide sequence was determined using a DNA sequencer (ALF DNA sequencer II, Pharmacia). As a result, a fragment highly homologous to the β subunit gene was obtained. This fragment is called ECL random prime labelling and detection sys
Using tem (Amersham), the probe was labeled with a random primer to obtain a probe. On the other hand, Southern blotting was performed using genomic DNA cut with HindIII.

The position of the 8 kbp band reacted with the above fragment
DNA was extracted from the gel, ligated to pUC18, and the nucleotide sequence of this DNA was determined. From the determined nucleotide sequence, chaperonin β
The start and stop codons of the subunit gene were located. The region containing the start codon is placed at the NdeI recognition site,
After modifying the region containing the stop codon at the BamHI recognition site using PCR, the region containing the ORF was replaced with the commercially available expression vector pET21a.
(Toyobo) to produce an expression plasmid pK1Eβ.

Each of the above expression plasmids was introduced into host E. coli BL21 (DE3), and cultured at 37 ° C. in 2 × YT (containing ampicillin or kanamycin). The cells were collected by centrifugation, and the buffer (50 mM Tris-HCl pH
7.5, 25 mM magnesium chloride) and sonicated. After removing the debris, the mixture was incubated at 70 ° C. for 30 minutes to precipitate and remove the denatured E. coli-derived protein. After adding ammonium sulfate to the supernatant to 30% saturation,
The mixture was adsorbed on a -toyopearl column (Tosoh) and eluted with a linear concentration gradient of 30% to 0%. The fractions containing the chaperonin protein were collected, dialyzed, applied to a Resource-Q column (Pharmacia), and eluted with a linear concentration gradient of 0 to 0.5 M NaCl. Collect the fraction containing the chaperonin protein and
Concentrate with triprep 30 (Amicon) and filter by gel (Shodex P
ROTEIN KW803, Showa Denko) and refined chaperonin β
Got a subunit.

Example 2 Stabilization of Green Fluorescent Protein by AMP-PNP-dependent β-chaperonin Homo-oligomer Green fluorescet Protein (hereinafter referred to as “GF”)
FIG. 1 shows the results of the thermal stabilization (abbreviated as “P”).
●). GFP is a jellyfish-derived protein that emits green fluorescence with a monomer having a molecular weight of about 27,000. GFP was denatured and diluted in a solution containing chaperonin, and a stabilization experiment was performed at 60 ° C. For denaturation of GFP, hydrochloric acid was added to the GFP solution at 0.0125 mM and DTT
Was added to a concentration of 5 mM, and the GFP concentration was adjusted to 10 μM for denaturation. Denatured GFP is
A regeneration buffer containing 50 or more β-chaperonin homo-oligomer heated to 0 ° C and equal to or more than GFP (50
mM Tris-HCl pH 7.0, 100 mM KCl, 25 mM MgCl ₂ , and 5
mM DTT). The folding of GFP was performed by applying excitation light of 396 nm and measuring fluorescence at 510 nm over time using a fluorimeter. When denatured GFP was diluted in a solution containing β-chaperonin homo-oligomer, the fluorescence of GFP was not recovered at all. This is presumably because the modified GFP was captured by the β-chaperonin homo-oligomer, so that the GFP could not form a normal three-dimensional structure. In the state where β-chaperonin homo-oligomer captures denatured GFP, AMP-P
Upon addition of NP, the fluorescence of GFP recovered rapidly and then remained constant. This indicates that β-chaperonin homo-oligomer and GFP captured by AMP-PNP are folded inside the chaperonin cavity and form a normal three-dimensional structure. In addition, since the fluorescence is constant, GFP is folded and protected as it is inside the chaperonin cavity, indicating that GFP is prevented from being denatured by heat or the like and is stabilized.

Comparative Example 1 The procedure of Example 2 was repeated except that AMP-PNP and β-chaperonin homo-oligomer were not added. The results are shown in FIG. 1 (FIG. 1, x). When diluted in a regeneration buffer without β-chaperonin homo-oligomer and AMP-PNP, fluorescence is generated rapidly. But,
Since it does not contain β-chaperonin homo-oligomer and AMP-PNP, it is not stabilized, and thermal denaturation of GFP occurs, and its fluorescence gradually decreases.

Example 3 Analysis of Stabilization of Green Fluorescent Protein by AMP-PNP-Dependent β-Chaperonin Homo-oligomer by Gel Filtration 100 μl of the regenerated solution reacted in the same manner as in Example 2 was collected.
It was subjected to gel filtration analysis. The result is shown in FIG. Gel filtration buffer was 50 mM Tris-HCl pH 7.0, 100 mM KCl,
The flow was set to 25 mM MgCl ₂ at a flow rate of 0.5 ml / min. The gel filtration column was analyzed using G3000SWxL (Tosoh) while heating and maintaining the temperature at 60 ° C. For protein detection, absorption at 280 nm of ultraviolet light (black line) and GFP fluorescence (398 nm of excitation light and 510 nm of fluorescence) (grey line) were measured in real time. As a result, the UV absorption peak corresponding to β-chaperonin homo-oligomer and GF
The behavior of P fluorescence peak coincided. This indicates that the captured GFP is retained inside the chaperonin cavity by the β-chaperonin homo-oligomer and AMP-PNP.

[Comparative Example 2] The same as Example 2 except that ATP was used instead of AMP-PNP. The results are shown in FIG. When ATP was used instead of AMP-PNP, the peak of UV absorption corresponding to β-chaperonin homo-oligomer and GF
The result that the fluorescence peak of P coincides is not obtained. This is G
This indicates that FP is not retained inside the cavity of the β-chaperonin homo-oligomer and is released into the solution.

Example 4 AMP-PN by Protease
Analysis of stabilization of green fluorescent protein by P-dependent β-chaperonin homo-oligomer Protease was added to the regenerated solution reacted in the same manner as in Example 2, and protease resistance was analyzed. The result is shown in FIG.
It was shown to. Protease is thermolysin (Wako Pure Chemical)
And a lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) at a concentration of 10 ng / μl.
For 10 minutes. Immediately after the reaction, add an equal volume of SDS-PAGE buffer and incubate at 95 ° C for 5 minutes.
It was subjected to 15% SDS-PAGE. After electrophoresis, the separated proteins were transferred to a PVDF membrane, and GFP was detected by Western blotting. First, the PVDF membrane was immersed in Block Ace (Dainippon Pharmaceutical) and left for 1 hour at room temperature. Next, PVDF membrane is anti-GF
It was immersed in a P rabbit antibody (Novagen) and left at room temperature for 1 hour. After standing, a PBS buffer (8.1 mM Na ₂ HPO ₄ , 137 mM
NaCl, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ), and washed three times for 10 minutes. Next, the PVDF membrane was immersed in horseradish peroxidase-labeled anti-rabbit IgG antibody (Bio-Rad), and left at room temperature for 1 hour. After standing, 1 in PBS buffer
Washed three times for 0 minutes. Antibody was used as a coloring solution (0.2 mg / ml of diamido
Nobenzidine, 50 mM Tris-HCl pH 7.2, 0.1% H ₂ O ₂ ) for staining.

The β-chaperonin homo-oligomer alone and β
When a chaperonin homo-oligomer and ATP were used (FIG. 4, lanes 1, 2, 5, and 6), the GFP band was thinned by protease treatment and degraded by the protease (FIG. 4, lanes 2 and 6). ). on the other hand,
When β-chaperonin homo-oligomer and AMP-PNP were contained (Fig. 4, lanes 3 and 4), GFP remained after protease treatment.
Band remains and has not been degraded by protease (FIG. 4, lane 4). This indicates that the captured GFP is retained inside the chaperonin cavity by the β-chaperonin homo-oligomer and AMP-PNP, and is stably present without being decomposed by the protease.

[0029]

According to the present invention, protein denaturation due to heat or the like can be suppressed, and the protein can function stably for a long period of time. In addition, it becomes possible to improve the heat resistance of a protein that is unstable to heat. By applying the present invention, regeneration of a denatured protein, stabilization of a protein reagent, enzymatic reaction at a high temperature, and the like can be performed.

[Brief description of the drawings]

FIG. 1 is a diagram showing the change over time in the fluorescence intensity of GFP.

FIG. 2: GFP, β-chaperonin oligomer, and AMP-PNP
FIG. 5 is a diagram showing the results of gel filtration analysis of a solution containing.

FIG. 3 shows the results of gel filtration analysis of a solution containing GFP, β-chaperonin oligomer, and ATP.

FIG. 4 is a diagram showing the results of electrophoresis when a protease is allowed to act on GFP under conditions where a β-chaperonin oligomer or the like is present.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Akira Ideno 751-1, Hirata, Kamaishi-shi, Iwate Pref. 3rd Land 75-1 Marine Corps Biotechnology Research Institute Kamaishi Laboratory F Term (Reference) 4B024 AA20 BA63 CA04 EA04 GA11 HA01 4H045 AA20 BA41 CA11 FA74 GA45

Claims (5)

[Claims] 1. A method for stabilizing a protein, comprising continuously retaining a native protein molecule inside a cavity of a group 2 type chaperonin molecule. 2. The method for stabilizing a protein according to claim 1, wherein the means for continuously retaining the native protein is to bind a nucleotide analog to a nucleotide reaction site of a group 2 type chaperone. 3. The nucleotide analogue is AMP-PNP, GMP
The method for stabilizing a protein according to claim 2, which is -PNP, CMP-PNP, or UMP-PNP. 4. The method for stabilizing a protein according to claim 1, wherein the group 2 type chaperonin is a chaperonin derived from archaebacteria. 5. The method for stabilizing a protein according to claim 1, wherein the group 2 type chaperonin is a chaperonin derived from a thermophilic archaeon or a hyperthermophilic archaeon.