JP4475826B2 - Protein stabilization method - Google Patents

Description

【００２０】
サーモコッカスsp. KS-1 株のDNAを鋳型としてPCR amplification kit （宝酒造）を用いたPCRを行った。条件は、初期変性94℃3分、ついで変性（94℃1分）、アニーリング（55℃1分）、伸長（72℃1分）のサイクルを30回行った。約800bpの増幅DNA断片をpUC18に連結し、コンピテントセルE. coli DH5αを用いて形質転換した。陽性クローンを選択し、挿入断片のあることを確認後、2×YT培地（0.05mg/ml のアンピシリンを含む）に植菌し、一晩37℃で振盪培養した。培養液を遠心分離して集菌後、プラスミドDNAを調製した。１サンプルあたり200ngのDNAを鋳型としてシークエンス反応を行い、DNAシークエンサー（ALF DNA sequencer II, ファルマシア）により塩基配列を決定した。その結果、βサブユニット遺伝子と相同性の高い断片が得られた。この断片をECL random prime labelling and detection system（アマシャム）を用いてランダムプライマーによりラベルし、プローブとした。一方、HindIIIで切断したゲノムDNAを用いてサザンブロッティングを行った。
【００２１】
上記断片と反応した8kbpのバンドの位置のDNAをゲルから抽出し、pUC18に連結し、このDNAの塩基配列を決定した。
決定した塩基配列からシャペロニンβサブユニット遺伝子の開始コドン及び終止コドンの位置を特定した。開始コドンを含む領域をNdeI認識部位に、終止コドンを含む領域をBamHI認識部位にPCRを用いて改変した後、ORFを含む領域を市販の発現ベクターpET21a（東洋紡）に組み込み、発現プラスミドpK1Eβを作製した。
【００２２】
上記発現用プラスミドをそれぞれ宿主大腸菌E. coli BL21(DE3)に導入し、２×YT（アンピシリンまたはカナマイシンを含む）で37℃で培養した。菌体を遠心分離によって回収し、緩衝液（50 mM Tris-HCl pH7.5, 25 mM 塩化マグネシウム）に懸濁し、超音波破砕を行った。debrisを除いたのち、70℃30分保温し、変性した大腸菌由来のタンパク質を沈殿除去した。その上澄に硫酸アンモニウムを30%飽和になるよう添加後、Ether-toyopearlカラム（東ソー）に吸着させ、30%〜0%の直線濃度勾配により溶出した。シャペロニンタンパク質を含む画分を集め、透析後 Resource-Qカラム（ファルマシア）にかけ、0〜0.5 MのNaClの直線濃度勾配により溶出した。シャペロニンタンパク質を含む画分を集め、Cetriprep 30（アミコン）で濃縮し、ゲル濾過（Shodex PROTEIN KW803、昭和電工）を行い、精製シャペロニンβサブユニットを得た。
【００２３】
〔実施例２〕 AMP-PNP依存のβシャペロニンホモオリゴマーによる緑色蛍光蛋白質の安定化
AMP-PNP依存のβシャペロニンホモオリゴマーによる緑色蛍光蛋白質（Green fluorescet Protein;以下、「GFP」と略す）の熱安定化の結果を図１に示した（図１，●）。GFPは、分子量約2万7千の単量体で緑色の蛍光を発するクラゲ由来の蛋白質である。GFPを変性させ、シャペロニンを含む溶液に希釈し、60℃での安定化実験を行った。GFPの変性は、GFP溶液に塩酸を0.0125mM、DTTを5mMとなるように添加し、GFP濃度は、10μMとなるように調製し変性させた。変性させたGFPは、あらかじめ60℃に加熱保温しておいたβシャペロニンホモオリゴマーをGFPと等量もしくはそれ以上含む再生バッファ（50 mM Tris-HCl pH 7.0, 100 mM KCl, 25 mM MgCl₂, and 5 mM DTT）に300倍希釈した。GFPの折り畳みは、蛍光光度計を用いて、励起光396nmをあて、蛍光510nmを径時的に追跡した。変性GFPを、βシャペロニンホモオリゴマーを含む溶液に希釈した場合、GFPの蛍光は全く回復されていない。これは、βシャペロニンホモオリゴマーにより変性GFPが捕捉された結果、GFPが正常な立体構造を形成できなくなったためと考えられる。βシャペロニンホモオリゴマーが変性GFPを捕捉している状態に、AMP-PNPを添加すると、GFPの蛍光は急速に回復し、その後一定に保たれた。これは、βシャペロニンホモオリゴマーとAMP-PNPにより捕捉されたGFPがシャペロニン空洞内部で折り畳まれ、正常な立体構造を形成していることを示している。また、蛍光が一定であることから、シャペロニン空洞内部にGFPが折り畳まれ、そのまま保護されているため、GFPは、熱などによる変性が防がれ、安定化されていることを示している。
【００２４】
〔比較例１〕
AMP-PNPとβシャペロニンホモオリゴマーを添加しなかったこと以外は実施例２と同様にした。その結果を図１に示した（図１，×）。βシャペロニンホモオリゴマーとAMP-PNPを含まない再生バッファに希釈した場合、急速に蛍光が生じる。しかし、βシャペロニンホモオリゴマー及びAMP-PNPを含まないため安定化されず、GFPの熱変性が生じその蛍光が少しづつ減少する。
【００２５】
〔実施例３〕 ゲル濾過によるAMP-PNP依存のβシャペロニンホモオリゴマーによる緑色蛍光蛋白質の安定化の解析
実施例２と同様に反応させた再生溶液を100μl分取し、ゲル濾過分析にかけた。その結果を図２に示した。ゲル濾過バッファは、50 mM Tris-HCl pH 7.0, 100 mM KCl, 25 mM MgCl₂とし、流速0.5ml/minで流した。ゲル濾過カラムはG3000SWxL（東ソー）を用い60℃に加熱保温しながら解析を行った。蛋白質の検出には、紫外光280nmの吸収（黒線）とGFP蛍光（励起光398nm蛍光510nm）（灰色線）をリアルタイムで測定した。その結果、βシャペロニンホモオリゴマーに相当する紫外吸収のピークとGFPの蛍光ピークが一致する挙動を示した。これは、βシャペロニンホモオリゴマーとAMP-PNPにより、捕捉されたGFPがシャペロニン空洞内部に保持されていること示している。
【００２６】
〔比較例２〕
AMP-PNPの代わりにATPを用いたこと以外は実施例２と同様である。結果を図３に示した。AMP-PNPの代わりにATPを用いた場合には、βシャペロニンホモオリゴマーに相当する紫外吸収のピークとGFPの蛍光ピークが一致する結果は得られない。これは、GFPがβシャペロニンホモオリゴマーの空洞内部に保持されず、溶液中に放出されることを示している。
【００２７】
〔実施例４〕 プロテアーゼによるAMP-PNP依存のβシャペロニンホモオリゴマーによる緑色蛍光蛋白質の安定化の解析
実施例２と同様に反応させた再生溶液にプロテアーゼを添加し、プロテアーゼ耐性を分析した。その結果を図４に示した。プロテアーゼは、サーモリシン（和光純薬）を1ng/μl濃度とリジルエンドペプチダーゼ（和光純薬）を10ng/μl濃度になるように再生溶液添加し、60℃で10分間反応させた。反応後、ただちにSDS-PAGEバッファを等量加え、95℃5分間インキュベートし、20μl分を15％SDS-PAGEにかけた。電気泳動後、分離された蛋白質はPVDF膜に転写し、GFPはウエスタンブロティングにより検出した。まず、PVDF膜をブロックエース（大日本製薬）に浸し、１時間室温で放置した。次にPVDF膜を抗GFPウサギ抗体（ノバジェン）に浸し、１時間、室温に放置した。放置後、PBSバッファ（8.1mM Na₂HPO₄, 137mM NaCl, 2.68mM KCl, 1.47mM KH₂PO₄）にて10分間3回洗浄した。次にホースラディッシュペルオキシダーゼラベルされた抗ウサギIgG抗体（バイオラッド）にPVDF膜を浸し、１時間室温に放置した。放置後、PBSバッファにて10分間3回洗浄した。抗体は発色液（0.2 mg/ml of diaminobenzidine, 50 mM Tris-HCl pH 7.2, 0.1% H₂O₂）に浸して染色した。
【００２８】
βシャペロニンホモオリゴマーのみ及びβシャペロニンホモオリゴマーとATPを用いた場合には（図４、レーン１、２、５、６）、プロテアーゼ処理するとGFPのバンドは薄くなり、プロテアーゼにより分解されてしまう(図４、レーン２及びレーン６）。一方、βシャペロニンホモオリゴマーとAMP-PNPを含む場合には（図４、レーン３、４）、プロテアーゼ処理後もGFPのバンドは残り、プロテアーゼによる分解がされていない（図４、レーン４）。これは、βシャペロニンホモオリゴマーとAMP-PNPにより、捕捉されたGFPがシャペロニン空洞内部に保持され、プロテアーゼによる分解を受けず、安定に存在していることを示している。
【００２９】
【発明の効果】
本発明により、熱などによるタンパク質の変性を抑制し、長期間タンパク質を安定に機能させることができる。また、熱に対して不安定なタンパク質の耐熱性を向上させることが可能となる。本発明を応用することで、変性タンパク質の再生、タンパク質試薬の安定化、高温での酵素反応などが可能となる。
【図面の簡単な説明】
【図１】 GFPの蛍光強度の経時的変化を示す図である。
【図２】 GFP、βシャペロニンオリゴマー、及びAMP-PNPを含む溶液のゲル濾過分析の結果を示す図である。
【図３】 GFP、βシャペロニンオリゴマー、及びATPを含む溶液のゲル濾過分析の結果を示す図である。
【図４】βシャペロニンオリゴマー等の存在する条件下で、GFPにプロテアーゼを作用させた場合の電気泳動の結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for preventing a natural protein from being denatured by environmental stress such as heat and keeping it in a stable state.
[0002]
[Prior art]
A protein is a polypeptide in which a plurality of amino acids are linked by peptide bonds. In order for a protein to express its properties, a unique tertiary structure (three-dimensional structure) formed by intramolecular or intermolecular interaction is important. In general, when a natural protein is subjected to environmental stress such as heat, its three-dimensional structure is changed and its properties are irreversibly lost (denatured protein). Therefore, how to maintain a natural protein against such environmental changes has always been a problem in handling proteins.
[0003]
As a solution to the above problems, intensive research has been conducted so far, and various improvements have been proposed. In recent years, interest has been increasing in molecular chaperones as factors involved in protein conformation and structural changes. Chaperonins, which are one of the molecular chaperones, are a group of heat shock proteins, and accumulate in cells when cells are exposed to various environmental stresses such as temperature changes. These are widely present regardless of eubacteria, archaea, and eukaryotes, and it has been clarified that they are involved non-specifically in the formation of a three-dimensional protein structure regardless of the type of protein. In particular, GroEL is well known as a chaperonin produced from eubacteria. For example, GroEL of Escherichia coli has a characteristic structure consisting of a total of 14 subunits in which donut-shaped structures in which 7 subunits are linked in a ring are 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 three-dimensional structure by hydrolysis of nucleotides such as ATP and binding of co-factor GroES. ing. Several attempts have been made to apply this chaperonin to protein stabilization. For example, Japanese Patent Application Laid-Open No. 7-67641 proposes a “method for stabilizing an enzyme in a solution by containing an enzyme-containing solution with nucleotides such as chaperonin protein and ATP”. Japanese Patent Laid-Open No. 7-48398 discloses that “an inactive protein that has been chemically denatured, an inactive protein accumulated in a transformant used in genetic manipulation, etc. is regenerated into an active protein. For this purpose, a method using a chaperonin purified from Thermus thermophilus has been proposed.
[0004]
All of these utilize the three-dimensional structure-forming effect of chaperonin, and when the three-dimensional structure is deformed by heat or denaturing agents, the protein chain of the protein is unwound (folded) back to its original three-dimensional structure. To achieve the purpose.
[0005]
However, chaperonins are generally ATP (adenosine-5 'triphosphate), CTP (cytidine-5' triphosphate), GTP (guanosine-5 'triphosphate), UTP (uridine-5' triphosphate) As the folding reaction proceeds with the nucleotide hydrolysis reaction, it is necessary to supply expensive nucleotides as needed to stabilize the protein. In addition, these nucleotides were easily decomposed by heat or pH change accompanying the hydrolysis reaction and lacked economic efficiency. Furthermore, in these methods, since the natural protein once folded by chaperonin is released from the chaperonin cavity, there is a risk of denaturation again.
[0006]
[Problems to be solved by the invention]
In view of the above problems, an object of the present invention is to provide a method for stabilizing a useful protein using chaperonin and supplying its characteristics effectively without supplying nucleotides as needed.
[0007]
[Means for Solving the Problems]
Usually, after the denatured protein is coupled with the hydrolysis of nucleotides and folded inside the chaperonin, it is released from the cavity as a natural form, and again, the protein is exposed to the risk of denaturation. However, as a result of voluntary studies by the present inventors, when a nucleotide analog was used instead of a nucleotide in the folding reaction, the denatured protein was folded in the chaperonin cavity by binding the nucleotide analog to the nucleotide reaction site of chaperonin, It was found that it was continuously trapped inside the chaperonin even after it was converted to the natural form, thus completing the present invention.
That is, the present invention is a protein stabilization method characterized in that a natural protein molecule is continuously retained inside a cavity of a group 2 type chaperonin molecule.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
Chaperonins are classified into two types, group 1 type derived from eubacteria and group 2 type derived from archaea or eukaryotes (Kim et al. Trends Biochem. Sci. 543-548, 1994). In the present invention, chaperonins belonging to group 2 type are used. Chaperonins classified into group 1 type require the binding of GroES, which is a 10 kDa subunit hexamer, as a cofactor for its folding reaction, as represented by GroEL derived from E. coli. When a protein denatured by heat or the like is folded by chaperonin, the denatured protein is trapped in the cavity of the donut structure of chaperonin. However, in the case of group 1 type chaperonin, GroES closes the cavity of chaperonin. When trapped inside, the enzyme and substrate molecules cannot react. On the other hand, group 2 type chaperonins, as represented by TF55, form oligomers only with subunits of 55-60 kDa and show folding activity without cofactors like GroES. Therefore, in the case of group 2 type chaperonins, since low molecular weight compounds can be transferred between the chaperonin cavities and the outside, the chaperonin cavities exhibit the intrinsic properties of proteins such as substance conversion in enzyme reactions. It becomes possible. Normally, when a group 2 type chaperonin captures a denatured protein and hydrolysis of nucleotides coexisting in the reaction solution, the quaternary structure of the chaperonin changes, and the unwound protein is released from the molecular cavity. . Continuous capture of the natural protein in the chaperonin cavity can be achieved by using homologues thereof instead of conventionally used nucleotides such as ATP, GTP, UTP, and CTP. Once the nucleotide homolog is bound to the nucleotide reaction site of the chaperonin molecule, it does not hydrolyze, so that the folded protein remains in the chaperonin cavity without any chaperonin structural change. Specifically, a denatured protein is captured by chaperonin in a solution, and a nucleotide homolog is added to perform a folding reaction inside the chaperonin. The nucleotide homologue binds to chaperonin but does not hydrolyze, so that the folded natural protein remains in the natural form in the chaperonin cavity and is not released to the outside.
[0009]
The nucleotide analog used in the present invention is one that binds to the nucleotide reaction site of chaperonin and does not dissociate. Examples include AMP-PNP (5'-adenylylimido-diphosphate), GMP-PNP (5'-guanylyimido-diphosphate), CNP-PNP (5'-cytidylylimido-diphosphate), UMP-PNP (5'-uridylylimido-diphosphate) Etc. are preferably used. Particularly desirable is AMP-PNP, but is not limited thereto.
[0010]
The chaperonins may be derived from eukaryotes, eubacteria and archaea, but the chaperonins used in the present invention may be any chaperonin of group 2 type chaperonins derived from archaea or eukaryotes. It is possible to use. The archaeal chaperonin has only 1-3 types of chaperonin monomers, so it is very convenient when producing chaperonin in large quantities by genetic recombination technology using E. coli. In particular, chaperonin derived from thermophilic or hyperthermophilic archaea is excellent in heat resistance of the chaperonin molecule itself, and thus the protein trapped by chaperonin can be 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 45-80 ° C., and the hyperthermophilic bacterium grows at 80 ° C. or higher.
[0011]
Archaea used in the present invention, Ashidianusu (Acidianus) genus Metarosufaera (Metallosphaera) genus, Sutiji Oro bus (Stygiolobus) genus Sulfolobus (Sulfolobus) genus, sul furo Lactococcus (Sulfurococcus) genus and Frith file gills (Sulfurisphaera) genus sul Roman Valles (Sulfolobales) eye, such as, Aeropairamu (Aeropyrum) genus, Death Le flow Lactococcus (Desulfurococcus) genus, Sutetteria (Stetteria) species, Staphylococcus Sir mass (Staphylothermus) genus, thermo discus (Thermodiscus) genus, IG neo Lactococcus ( Igneococcus) genus, Thermos file gills (Thermosphaera) genus and Fofo ball Lactococcus (Sulfophobococcus) genus, hyper Sir mass (Hyperthermus) genus, Pyro decrease-tee Umm (Pyrodictium) genus and Pairorobasu (Pyrolobus) species such as IG neo Cocca-less (Igneococcules ), Pyrobaculum genus, Thermop Roteusu (Thermoproteus) genus, thermophilum (Thermofilum) genus and Cardo Lactococcus (Caldococcus) genus Thermo Protea less, such as (Thermoproteales) eyes, Akiogurobusu (Archaeoglobus) genus and Ferogurobusu (Ferroglobus) genus archetypes og ass-less, such as (Archaeoglobales) eyes, Metanosamasu (Methanotherm us) genus, methanolate Agrobacterium (Methanobacterium) genus, methanolate thermo Enterobacter (Methanothermobacter) genus and Metanosufaera (Methanosphaera) genus methanolate bacteria less (Methanobacteriales) eye, such as, Methanococcus (Methanococcus) genus, methanolate Thermococcus (Methanothermococcus) genus, methanolate cardo Lactococcus (Methanocaldococcus) genus and Metanoigunisu (Methanoignis) Metanokokkaresu (Methanococcales) eye, such as the genus, methanolate micro vias less (Methanomicrobiales) eyes, Metanozaruchina (Methanosarcina) Metanozaruchina such as the genus Scan (Methanosarcinales) eyes, Metanopairaresu (Methanopyrales) eyes, Pyrococcus (Pyrococcus) genus and Thermococcus (Thermococcus) genus Thermo Cocca-less (Thermococcales) eye, such as, Thermo plasma (Thermoplasma) genus and Pikurofirasu (Picrophilus) species such as such as thermo plus Mares (Thermoplasmales) eyes, and the like. The chaperonin used in the present invention may be derived from any archaebacteria, and is not limited thereto, but among these, those derived from thermophilic or hyperthermophilic archaea are preferred.
[0012]
Since the protein that can be stabilized in the present invention needs to be trapped in the cavity of the chaperonin molecule, its molecular weight is desirably about 60 kDa or less.
In order to obtain the chaperonin used in the present invention, a thermophilic archaebacterium or a hyperthermophilic archaebacterium is cultured, the resulting microbial cells are lysed using a bacterium solubilizing agent such as SDS, phenol extraction and ethanol precipitation. Genomic DNA is extracted by techniques such as the method. This genomic DNA is cleaved with an appropriate restriction enzyme and then ligated to an appropriate vector to prepare a genomic DNA library. Various vectors derived from λ phage, for example, phagemid DNA such as λgt10 and λZAP, or plasmid vectors such as pUC18 and pBR322 can be used as the vector. On the other hand, based on the amino acid sequence of a region having high homology between chaperonin genes derived from other species, a corresponding DNA is synthesized and used as a primer for PCR. A partial fragment of the target gene can be obtained by PCR using the genomic DNA as a template using this primer. The partial fragment can be used as a gene screening probe by labeling with a radioactive element such as [ ³² P] or a non-radioactive compound such as dicoxygenin.
[0013]
In order to obtain the entire nucleotide sequence of the target gene, the genomic DNA library is introduced into a host such as E. coli, and a clone that binds strongly to the labeled probe is selected. The base sequence can be determined by a general method such as the Sanger method or the Maxam-Gilbert method. Through the above procedure, the entire DNA base sequence encoding chaperonin including the stop codon can be isolated from the translation start codon. By the above operation, the isolated chaperonin-encoding DNA can be appropriately inserted into an expression vector such as a pET system, and introduced into microorganisms or cultured cells to be expressed, whereby a large amount of the desired chaperonin can be prepared.
[0014]
In order to capture the denatured protein in the chaperonin, the chaperonin oligomer may be mixed with the denatured protein 1 at a mixing ratio of 0.1-10 in the solution. Preferably, the mixing ratio of chaperonin oligomer 0.5-2 to 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. The concentration range of potassium ions is preferably 10-1000 mM, and the concentration of magnesium ions is preferably 10-500 mM.
[0015]
After capturing the denatured protein with chaperonin, the protein captured in the chaperonin cavity is folded into a natural form by adding a nucleotide analog, but it is not released outside the chaperonin as in the case of adding a nucleotide. The amount of nucleotide analog added is preferably 0.1-10 mM.
[0016]
The natural protein captured in the group 2 type chaperonin cavity is not isolated from the outside as in the case of being captured by the group 1 type chaperonin, and the mass transfer between the cavity and the outside is possible. Therefore, it can be used in the same way as a normal protein. Since chaperonins obtained from thermophilic archaea and hyperthermophilic archaea are excellent in heat resistance, natural proteins trapped inside are also given heat resistance. Therefore, not only can the reaction be performed under a stress environment such as a high temperature, but it is advantageous in transportation and storage.
[0017]
【Example】
Hereinafter, the present invention will be described by way of examples, but the scope of the present invention is not limited thereto.
[Example 1] Preparation of chaperonin of hyperthermophilic archaeon β chaperonin homooligomer derived from the hyperthermophilic archaeon Thermococcus sp. KS-1 strain was prepared according to the method described in JP-A-10-327869.
[0018]
0.6 g of cells of Thermococcus sp. KS-1 strain was suspended in 10 ml of TNE buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM EDTA). 1 ml each of 10% SDS solution and 1% Triton X-100 solution was added and left overnight at 4 ° C. Subsequently, the temperature was adjusted to 50 ° C., 0.05 ml of proteinase K solution (20 mg / ml) was added, and the mixture was shaken for 4 hours. After phenol treatment and chloroform treatment, 0.05 ml of RNase A solution (0.5 mg / ml) was added and left at 37 ° 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 left at 50 ° C. for 70 minutes. After phenol treatment and chloroform treatment (solution volume: 10 ml), 1 ml of 3M sodium acetate solution (pH 5.2) and 25 ml of ethanol were added and left at -20 ° C. for 2 hours. Thereafter, the mixture was centrifuged with a high-speed centrifuge to precipitate DNA, washed with 3 ml of 70% ethanol solution, dried to dryness with a centrifugal evaporator, and dissolved in 0.5 ml of TE buffer. By this operation, about 2 mg of genomic DNA was obtained.
[0019]
The following DNA primers were synthesized from the amino acid homology region of archaeal chaperonin.

[0020]
PCR was performed using PCR amplification kit (Takara Shuzo) using DNA of Thermococcus sp. KS-1 as a template. The conditions were initial denaturation 94 ° C. for 3 minutes, followed by 30 cycles of denaturation (94 ° C. for 1 minute), annealing (55 ° C. for 1 minute), and extension (72 ° C. for 1 minute). An about 800 bp amplified DNA fragment was ligated to pUC18 and transformed using competent cell E. coli DH5α. After selecting a positive clone and confirming the presence of the inserted fragment, it was inoculated into 2 × YT medium (containing 0.05 mg / ml ampicillin) and cultured overnight at 37 ° C. with shaking. The culture solution was centrifuged and collected, and then 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 with a DNA sequencer (ALF DNA sequencer II, Pharmacia). As a result, a fragment having high homology with the β subunit gene was obtained. This fragment was labeled with a random primer using ECL random prime labeling and detection system (Amersham), and used as a probe. On the other hand, Southern blotting was performed using genomic DNA cut with HindIII.
[0021]
The DNA at the position of the 8 kbp band reacted with the above fragment was extracted from the gel and ligated to pUC18, and the base sequence of this DNA was determined.
From the determined base sequence, the positions of the start codon and stop codon of the chaperonin β subunit gene were identified. After modifying the region containing the start codon into the NdeI recognition site and the region containing the stop codon into the BamHI recognition site using PCR, the region containing the ORF was incorporated into the commercially available expression vector pET21a (Toyobo) to produce the expression plasmid pK1Eβ did.
[0022]
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, suspended in a buffer solution (50 mM Tris-HCl pH 7.5, 25 mM magnesium chloride), and subjected to ultrasonic disruption. After removing debris, the mixture was kept at 70 ° C. for 30 minutes to precipitate and remove the denatured E. coli-derived protein. Ammonium sulfate was added to the supernatant so as to be 30% saturated, then adsorbed on an Ether-toyopearl column (Tosoh), and eluted with a linear concentration gradient of 30% to 0%. Fractions containing chaperonin protein were collected, applied to a Resource-Q column (Pharmacia) after dialysis, and eluted with a linear gradient of 0-0.5 M NaCl. Fractions containing chaperonin protein were collected, concentrated with Cetriprep 30 (Amicon), and subjected to gel filtration (Shodex PROTEIN KW803, Showa Denko) to obtain purified chaperonin β subunit.
[0023]
[Example 2] Stabilization of green fluorescent protein by AMP-PNP-dependent β chaperonin homo-oligomer
The results of thermal stabilization of green fluorescet protein (hereinafter abbreviated as “GFP”) by an AMP-PNP-dependent β-chaperonin homooligomer are shown in FIG. 1 (FIG. 1, ●). GFP is a jellyfish-derived protein that emits green fluorescence with a molecular weight of about 27,000 monomers. GFP was denatured, diluted in a solution containing chaperonin, and a stabilization experiment at 60 ° C. was performed. To denature GFP, hydrochloric acid was added to the GFP solution at 0.0125 mM and DTT at 5 mM, and the GFP concentration was adjusted to 10 μM and denatured. The denatured GFP is a regeneration buffer (50 mM Tris-HCl pH 7.0, 100 mM KCl, 25 mM MgCl ₂ , and) containing a β-chaperonin homooligomer that has been heated and kept at 60 ° C. (5 mM DTT) was diluted 300 times. Folding of GFP was followed by chronologically tracking 510 nm of fluorescence by applying excitation light of 396 nm using a fluorometer. When denatured GFP is diluted in a solution containing β-chaperonin homooligomer, the fluorescence of GFP is not recovered at all. This is thought to be because GFP cannot form a normal three-dimensional structure as a result of capturing denatured GFP by β-chaperonin homo-oligomer. When AMP-PNP was added while β-chaperonin homo-oligomer was capturing denatured GFP, the fluorescence of GFP rapidly recovered and then kept constant. This indicates that the β chaperonin homooligomer and GFP captured by AMP-PNP are folded inside the chaperonin cavity to form a normal three-dimensional structure. In addition, since the fluorescence is constant, GFP is folded inside the chaperonin cavity and protected as it is, which indicates that GFP is stabilized by preventing denaturation due to heat and the like.
[0024]
[Comparative Example 1]
The procedure was the same as Example 2 except that AMP-PNP and β-chaperonin homooligomer were not added. The results are shown in FIG. 1 (FIG. 1, x). When diluted in a regeneration buffer that does not contain β-chaperonin homooligomer and AMP-PNP, fluorescence rapidly occurs. However, since it does not contain β-chaperonin homo-oligomer and AMP-PNP, it is not stabilized, and heat denaturation of GFP occurs and its fluorescence gradually decreases.
[0025]
[Example 3] Analysis of stabilization of green fluorescent protein by AMP-PNP-dependent β-chaperonin homooligomer by gel filtration 100 μl of the regenerated solution reacted in the same manner as in Example 2 was collected and subjected to gel filtration analysis. The results are shown in FIG. The gel filtration buffer was 50 mM Tris-HCl pH 7.0, 100 mM KCl, 25 mM MgCl ₂ and flowed at a flow rate of 0.5 ml / min. The gel filtration column was analyzed using G3000SWxL (Tosoh) while heating at 60 ° C. For protein detection, absorption at 280 nm in ultraviolet light (black line) and GFP fluorescence (excitation light at 398 nm fluorescence at 510 nm) (gray line) were measured in real time. As a result, the UV absorption peak corresponding to β chaperonin homo-oligomer and the fluorescence peak of GFP coincided. This indicates that the captured GFP is retained inside the chaperonin cavity by β-chaperonin homooligomer and AMP-PNP.
[0026]
[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 is used instead of AMP-PNP, a result in which the ultraviolet absorption peak corresponding to the β chaperonin homo-oligomer and the fluorescence peak of GFP coincide is not obtained. This indicates that GFP is not retained inside the cavity of the β chaperonin homooligomer and is released into the solution.
[0027]
[Example 4] Analysis of stabilization of green fluorescent protein by AMP-PNP-dependent β-chaperonin homooligomer by protease Protease was added to a regenerated solution reacted in the same manner as in Example 2, and protease resistance was analyzed. The results are shown in FIG. As the protease, thermolysin (Wako Pure Chemical Industries) was added to a regenerating solution so as to have a concentration of 1 ng / μl and lysyl endopeptidase (Wako Pure Chemical Industries) to a concentration of 10 ng / μl, and reacted at 60 ° C. for 10 minutes. Immediately after the reaction, an equal volume of SDS-PAGE buffer was added, incubated at 95 ° C. for 5 minutes, and 20 μl was subjected to 15% SDS-PAGE. After electrophoresis, the separated protein was 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 at room temperature for 1 hour. Next, the PVDF membrane was immersed in an anti-GFP rabbit antibody (Novagen) and left at room temperature for 1 hour. After standing, it was washed 3 times for 10 minutes with PBS buffer (8.1 mM Na ₂ HPO ₄ , 137 mM NaCl, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ). Next, the PVDF membrane was immersed in horseradish peroxidase-labeled anti-rabbit IgG antibody (BioRad) and left at room temperature for 1 hour. After standing, it was washed 3 times with PBS buffer for 10 minutes. The antibody was stained by soaking in a chromogenic solution (0.2 mg / ml of diaminobenzidine, 50 mM Tris-HCl pH 7.2, 0.1% H ₂ O ₂ ).
[0028]
When β-chaperonin homooligomer alone or β-chaperonin homooligomer and ATP are used (FIG. 4, lanes 1, 2, 5, and 6), the GFP band becomes thin when protease treatment is performed and is degraded by protease (FIG. 4, Lane 2 and Lane 6). On the other hand, when β-chaperonin homooligomer and AMP-PNP are contained (FIG. 4, lanes 3 and 4), a GFP band remains even after the protease treatment and is not degraded by protease (FIG. 4, lane 4). This indicates that the captured GFP is retained inside the chaperonin cavity by the β chaperonin homooligomer and AMP-PNP, and is stably present without being degraded by protease.
[0029]
【The invention's effect】
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. It also becomes possible to improve the heat resistance of proteins that are unstable to heat. By applying the present invention, it is possible to regenerate denatured proteins, stabilize protein reagents, enzymatic reactions at high temperatures, and the like.
[Brief description of the drawings]
FIG. 1 is a graph showing changes in fluorescence intensity of GFP over time.
FIG. 2 is a diagram showing the results of gel filtration analysis of a solution containing GFP, β-chaperonin oligomer, and AMP-PNP.
FIG. 3 is a diagram showing 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 protease is allowed to act on GFP under conditions where β-chaperonin oligomers and the like are present.

Claims (4)

Using β chaperonin homo-oligomers and AMP-PNP from archaea, the β chaperonin method for stabilizing a protein characterized Rukoto the cavity inside the homo-oligomer is continuously maintained the native protein molecule. The method for stabilizing a protein according to claim 1 , wherein the β-chaperonin homooligomer derived from archaea is a β-chaperonin homooligomer derived from thermophilic archaea or hyperthermophilic archaea. The protein stabilization method according to claim 1 or 2, wherein the protein stabilization is stabilization against thermal denaturation of the protein. The method for stabilizing a protein according to claim 1 or 2, wherein the stabilization of the protein is stabilization against enzymatic degradation of the protein.

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