JPH11266865A - Chaperonin oligomer originated from archaebacterium, its production and preservation, and regeneration of protein with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing chaperonin oligomer originated from an archaebacterium, capable of converting chaperonin monomer into the chaperonin oligomer, an active chaperonin useful for the regeneration, stabilization, heat stabilization, etc., of a useful protein, by treating the chaperonin monomer originated from the archaebacterium with magnesium ion and a nucleotide. SOLUTION: This method for producing chaperonin oligomer comprises treating chaperonin monomer originated from an archaebacterium with magnesium ion (MgCl2 , etc.), and a nucleotide (adenosine triphosphate, etc.), to produce the chaperonin oligomer. The chaperonin oligomer is preserved and maintained in the presence of the magnesium ion and the nucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an archaeal chaperonin oligomer, a method for producing and storing the same, and a method for regenerating a protein using the same. Chaperonin is an in vivo factor involved in protein folding and stabilization, and is expected to be used as a stabilizer for other coexisting useful proteins and to be applied to the regeneration process of useful proteins produced by genetic recombination technology. ing.

[0002]

2. Description of the Related Art Chaperonin is known as a protein folding factor, and has a function of improving the heat resistance and regeneration of a protein in combination with its ATP degrading activity. This function of chaperonin can be used for regeneration, stabilization, heat resistance and the like of useful proteins. For example, JP-A-7-67
No. 641 proposes a method for stabilizing an enzyme by adding nucleotides and chaperonin to an enzyme in a solution and utilizing a specific interaction between the enzyme, nucleotide and chaperonin.

Active chaperonin is an oligomer composed of subunits (monomers) having a molecular weight of about 60,000.
Each subunit forms a two-layered cyclic structure (Protein
science, 1994, 3 1436-1443, Bioscience and Industry Vol.55 No.9 (1997)). Therefore, in order to effectively utilize the function of chaperonin, a technique for efficiently converting a chaperonin monomer into an active chaperonin oligomer and maintaining the oligomer structure is required.

[0004]

However, it has been difficult with the conventional method to efficiently activate archaeal chaperonin. An object of the present invention is to solve the problems of the conventional method and to provide a technique for efficiently producing and storing a chaperonin oligomer.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, it has been found that chaperonin monomers derived from archaebacteria can be easily converted to chaperonin oligomers by treating them with magnesium ions and nucleotides. And completed the present invention. That is, the present invention provides a chaperonin monomer derived from archaebacteria,
A method for producing a chaperonin oligomer derived from an archaebacteria, wherein the chaperonin oligomer is treated with a magnesium ion and a nucleotide to obtain a chaperonin oligomer.

[0006] The present invention is also an archaeal chaperonin oligomer produced by the method described above. Furthermore, the present invention is a method for storing archaeal chaperonin oligomers, comprising storing archaeal chaperonin oligomers in the presence of magnesium ions and nucleotides. Furthermore, the present invention is a method for regenerating a protein, which comprises regenerating a protein using the archaeal chaperonin oligomer described above.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. (1) Method for Producing Chaperonin Oligomer The method for producing an archaeal chaperonin oligomer of the present invention is characterized in that an archaeal chaperonin monomer is treated with magnesium ions and nucleotides. Examples of the chaperonin monomer include, for example, Sulfolobus, Desulfurococcus and the like.
ococcus, Pyrodictium, Thermofilum, Thermoproteus (T
hermoproteus), Pyrobaculum
Genus, Pyrococcus, Methanobacterium, Methanococcus
occus, genus Methanopyrus, genus Methanothermus, and archablobs (Ar
chaeoglobus), Halobacterium
Genus, Methanoplanus, Methanospirillum, Methanosarcia
anosarcina) can be used, and among these, Methanococcus thermolystroficus (Methanoccus)
Preference is given to using chaperonin oligomers from B. occus thermolithotrophicus.

The chaperonin monomer can be obtained by introducing and expressing a chaperonin gene derived from archaebacteria using Escherichia coli or the like as a host, and purifying the recombinant chaperonin by ion exchange, column chromatography by hydrophobic interaction, or the like. The recombinant chaperonin thus obtained contains a mixture of monomers and oligomers, but is preferably treated with a metal chelating reagent such as ethylenediaminetetraacetic acid / disodium, and then prepared in advance with a buffer solution consisting of only a buffer. It is preferable that the recombinant chaperonin oligomer is once dissociated into monomers by dialysis. However, this dissociation into monomers may be performed in any purification step.

The treatment with magnesium ions and nucleotides can be performed by adding a chaperonin oligomer to a solution containing magnesium ions and nucleotides and reacting for a certain period of time. As a source of magnesium ions, magnesium chloride, magnesium sulfate, or the like can be used. The concentration of magnesium ion in the solution is not particularly limited, but is preferably 1 mM to 1000 mM.

As nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP), cytidine triphosphate
(CTP), uridine triphosphate (UTP), guanosine triphosphate
(GTP) can be used, but ATP is preferably used. The nucleotide concentration is preferably 0.01 mM to 10 mM, but is preferably at least the same molar amount as the number of subunit moles of the chaperonin oligomer. The solution preferably contains about 0.05-1.0 M ammonium sulfate or about 0.05-0.5 M KCl.
It is desirable to coexist. As a result, the oligomer production efficiency can be dramatically improved.

The reaction temperature and the reaction time are not particularly limited, but the temperature during the reaction is preferably between room temperature and 60 ° C., and the reaction time is preferably about 5 hours. Confirmation of oligomer formation can be confirmed by gel filtration or observation with an electron microscope. Reconstituted chaperonin,
Alternatively, after condensation by ultrafiltration or the like, stable storage is possible. The chaperonin derived from archaebacteria obtained in this manner is considered to have lower specificity for the target protein than the chaperonin derived from eubacteria, and to be superior in versatility. This is because the types of archebacterial protein folding factors are less than those of eubacteria such as Escherichia coli, so that protein folding is expressed with a minimum of types of folding factors.

(2) Method for Preserving Chaperonin Oligomers The method for storing archaeal chaperonin oligomers of the present invention is characterized by storing chaperonin monomers in the presence of magnesium ions and nucleotides. The kind of archaebacteria, the source and concentration of magnesium ion, the nucleotide and the concentration thereof may be the same as in the above-mentioned production method, and the preservation solution contains about 0.05-1.0M ammonium sulfate or 0.05-1.0M. It is preferable that about 0.5 M of KCl coexist. The temperature during storage is not particularly limited, but it is preferable to store at a low temperature such as -20 to 10 ° C.

(3) Method for Regenerating Protein The method for regenerating a protein of the present invention is characterized by using an archaeal chaperonin oligomer. More specifically, the method is characterized in that a denatured protein is added to a solution containing a chaperonin oligomer derived from an archaebacteria and reacted for a certain period of time to regenerate the denatured protein. The concentration of the chaperonin oligomer in the reaction solution is not particularly limited, but may be 0.1.
The concentration is preferably 1 to 1 μM. In addition, the kind of archaea used as a source of chaperonin may be the same as in the above-mentioned production method. Although the concentration of the denatured protein in the reaction solution is not particularly limited, it is preferably 0.1 to 5 μM. It is preferable to add ATP, potassium ion and magnesium ion to the reaction solution in addition to the chaperonin oligomer. Their concentrations are not particularly limited, but are each 0.
It is preferably 1 to 5 mM, 1 to 500 mM, 0.1 to 100 mM. The reaction temperature and reaction time are not particularly limited, but the temperature during the reaction is preferably 15 to 80 ° C, and the reaction time is preferably 0.05 to 120 hours.

[0014]

The present invention will be described below with reference to examples, but the scope of the present invention is not limited to these examples. [Example 1] Mesanococcus thermolystrophycus
An expression plasmid pETTS1 containing a chaperonin gene derived from the DSM2095 strain was introduced into Escherichia coli (this Escherichia coli has been deposited with the National Institute of Bioscience and Human Technology as FERM P-16438), and this was transferred to an LB medium (containing ampicillin). At 37 ℃
And cultured. When the OD650 of the culture reaches 1.5, 1m
M IPTG was added to induce the expression of the chaperonin gene. Thereafter, the culture was continued for 3 hours, and after the cells were collected by centrifugation, a buffer solution (50 mM Tris-HCl pH 7.5, 5 mM MgCl 2
Was suspended in ₂₎ was subjected to ultrasonic treatment for this suspension. After removing the crushed bacterial cells from the treatment solution, the mixture was incubated at 65 ° C. for 90 minutes to precipitate and remove the denatured Escherichia coli-derived protein. (NH ₄ ) ₂ SO ₄ was added to the obtained supernatant to a concentration of 1.3 M, and after ammonium sulfate salting out, the supernatant was washed with TSKgel Ether-5PW.
Purified by hydrophobic chromatography according to. The fraction containing a protein having a size corresponding to chaperonin was collected and dialyzed against 25 mM HEPES-KOH (pH 6.8), 5% glycerol buffer. TSKgel SuperQ-5PW
Purified by anion exchange chromatography on a column. After collecting a fraction containing a protein having a size corresponding to chaperonin, the fraction was concentrated by ultrafiltration, and the concentrate was subjected to gel filtration using a TSKgel G3000SWXL column (flow rate: 0.5 ml / min, developing solution: 25 mM HEPES-KOH).
(pH 6.8), 25 mM MgCl ₂ 5% Glycerol) to obtain a fraction containing chaperonin.

The obtained fraction of chaperonin was 10
3 mM mM EDTA (ethylenediaminetetraacetic acid, disodium)
After the treatment for 0 minutes, dialysis was performed at 25 ° C. for 24 hours at 4 ° C. against a HEPES-KOH buffer solution (pH 6.8). 50 mM MgC in dialysate
l ₂ , 5 at 30 ° C. in the presence of 0.25 mM ATP and 0.3 M (NH ₄ ) ₂ SO ₄
After time treatment, the chaperonin oligomer was reconstituted. After the treatment, a part was subjected to gel filtration using a TSKgelG3000SWXL column (Tosoh Corporation), and observation of the formation of chaperonin oligomers was observed by electron microscope observation. A 25 mM HEPES-KOH buffer (pH 6.8) containing 50 mM MgCl ₂ , 0.25 mM ATP, and 0.3 M (NH ₄ ) ₂ SO ₄ was used as a developing solution for gel filtration. After the reconstitution treatment, a peak likely to be a chaperonin oligomer appeared immediately after the elution amount exceeded the void volume by gel filtration (FIG. 1). The fraction showing this peak was analyzed by SDS-PAGE, and as a result, it was found to be chaperonin (FIG. 2). Further, as a result of observing the structure with an electron microscope, it was revealed that the structure had a ring structure unique to chaperonin (FIG. 3). This confirmed reconstitution into a chaperonin oligomer.

Example 2 The chaperonin oligomer solution obtained in Example 1 was stored at a concentration of 2 mg / ml at 4 ° C. for 30 days, and examined by observation with an electron microscope. As a result, in addition to the free oligomer, the chaperonin oligomer was polymerized via the ring surface to form a filament structure (FIG. 4). However, this filament was dissociated by diluting it below 0.5 mg / ml, which is the practical concentration of the chaperonin oligomer solution, even in the presence of magnesium ions and ATP. Therefore, this filament was confirmed to be a reversible polymerization.

Example 3 The ATPase activity of the chaperonin oligomer and the chaperonin monomer obtained in Example 1 was measured. ATPase activity is 50 mM HEPES-KOH, 50 mM M
To a test solution consisting of gCl ₂ , 300 mM KCl, and 2 mM ATP, 12.5, 25.0, or 50.0 μg of chaperonin oligomer or 50 μg of chaperonin monomer was added, and the temperature was measured at 65 ° C. As a result, concentration-dependent ATP degradation was observed for the chaperonin oligomer (FIG. 5), but no clear ATP degradation was observed for the chaperonin monomer. In addition, as a result of measuring the ATPase activity of the chaperonin oligomer in the range of 40-90 ° C, the optimal temperature was 60-70 ° C.
(FIG. 6).

Example 4 Citrate synthase (Sigma) derived from the thermophilic archaeon Thermoplasma acidophilum was added to a 50 mM HEPES-KOH buffer (pH 7.5) containing 6 M guanidium hydrochloride. , 50 ℃
For 30 minutes to denature the enzyme. Then, Example 1
50 mM HEPES-KOH
The regeneration reaction was started by adding a buffer (pH 7.5) and diluting 60-fold, and the enzymatic activity of citrate synthase was measured over time. For activity measurements, see Acta Chemica Scandina
vica, 1963, 17, S129-134 ".

The regeneration reaction is performed when ATP (final concentration: 2 mM) is contained from the beginning, or 10/20 minutes after the start of the reaction, and 1/20 during the reaction.
Two procedures were performed, one in which 0 volume of 400 mM ATP was added. As a control, the activity was also measured in the case where no chaperonin oligomer was added. All in each reaction
Include 300 mM KCl and 50 mM MgCl ₂ and measure
Performed at 50 ° C. The final concentrations of the citrate synthase and the chaperonin oligomer in the regeneration reaction were both 0.33 μM.

As a result, when chaperonin and ATP were present from the start of the reaction, the activity yield was dramatically increased as compared with spontaneous regeneration (FIG. 7). In addition, it was found that chaperonin inhibited the regeneration reaction in the absence of ATP, but accelerated the regeneration reaction after ATP was added. From this fact, the same action as Escherichia coli GroE that an archaeal chaperonin composed of a single subunit obtained by the present invention binds to a regeneration intermediate in the absence of ATP and releases it in the presence of ATP. It turned out to be effective.

[0021]

The chaperonin monomer derived from archaebacteria is
A method for efficiently converting and maintaining an active chaperonin oligomer is provided.

[Brief description of the drawings]

FIG. 1 shows the results of gel filtration of a reconstituted chaperonin.

FIG. 2 is a view showing a result of SDS-electrophoresis of a fraction expected to contain a chaperonin oligomer.

FIG. 3 is an electron micrograph of a chaperonin oligomer.

FIG. 4 is an electron micrograph of a chaperonin oligomer having a filament structure.

FIG. 5: Amount of added chaperonin oligomer and ATP
It is a figure which shows the relationship with activity.

FIG. 6 is a graph showing the relationship between temperature and ATP activity of a chaperonin oligomer.

FIG. 7 is a graph showing the relationship between the presence or absence of a chaperonin oligomer and the activity of citrate synthase.

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 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tadashi Maruyama 75-1 Hirata, Kamaishi-shi, Iwate Pref.

Claims (6)

[Claims] 1. An archaeal chaperonin monomer,
A method for producing a chaperonin oligomer derived from an archaebacteria, wherein the chaperonin oligomer is treated with a magnesium ion and a nucleotide to obtain a chaperonin oligomer. 2. The method for producing an archaeal chaperonin oligomer according to claim 1, wherein the nucleotide is adenosine triphosphate. 3. A chaperonin oligomer derived from archaebacteria produced by the method according to claim 1 or 2. 4. A method for storing archaeal chaperonin oligomers, comprising storing archaeal chaperonin oligomers in the presence of magnesium ions and nucleotides. 5. The method for preserving an archaeal chaperonin oligomer according to claim 4, wherein the nucleotide is adenosine triphosphate. 6. A method for regenerating a protein, comprising regenerating a protein using the archaeal chaperonin oligomer according to claim 3.