JP3934066B2 - Novel protein that forms spherical particles, and novel gene encoding the protein - Google Patents

Description

【０１２４】
表１の２番及び３番に示すSulfolobus属のタンパク質では、アミノ酸配列の長さ（アミノ酸残基数）がほとんど変わらず（約３４０アミノ酸残基）、類似性は約３０％であった。表１の４番及び５番に示すタンパク質は、同じPyrococcus属のタンパク質であり、タンパク質の長さが１０１残基と短いものの、５０％以上という高い類似性を示した。なお、上記４種以外のタンパク質は、タンパク質の長さが異なり、類似性も２０％を切るので、相同性があるとは言えないと判断した。
【０１２５】
図２０には、表１の２番に示すSulfolobus tokodaii ST 0992のアミノ酸配列と、P.furiosusのアミノ酸配列との比較結果を示している。表１に示すように類似性は３０％であるが、図２０によれば、前半部分の類似性のほうが、後半部分の類似性よりも高いことが分かる。
【０１２６】
なお、図２０と後述の図２１とにおいて、相同性を示す記号として、「＊」と「：」と「．」と「 」（空白）とを用いている。「＊」は、生物種間において、完全にアミノ酸配列が保存されていることを示している。「：」は、生物種間において、性質が非常に近いアミノ酸残基間の変異が起きていることを示している。「．」は、生物種間において、性質が近いアミノ酸残基間の変異が起きていることを示している。「 」（空白）は、生物種間において、「：」及び「．」で表すことができない変異が起きていることを示している。
【０１２７】
バクテリオファージＨＫ９７の場合は、立体構造が類似している部分があるので、類似性は相当高いと予想された。しかし、実際は、表１に示すように、アミノ酸配列の類似性が１９％とそれほど高くなかった。このことから、殻タンパク質は、それほど類似性がなくとも、ウイルス様粒子となる可能性が十分にあるということが推測される。
【０１２８】
相同性配列で見つかった４つのタンパク質（表１における番号２〜５のタンパク質）は、いずれも機能未知なタンパク質である。表１の番号２〜３に示すタンパク質の種であるSulfolobus属は、Crenarchaeota（古細菌のサブドメインの一つ）に分類される好酸性好熱菌で、１００℃以上でも生育する超好熱菌ではなく、P.furiosusと近い関係ではない。しかし、この目的のタンパク質（表１の番号２〜３に示すタンパク質）の類似性は、全体の約３０％の類似性をもつ。この数字は、表１に示すように、ＨＫ９７の殻タンパク質との類似性より高い。さらに、表１の番号２〜３に示すタンパク質のアミノ酸残基数は、どちらも、発明者の解析したウイルス様粒子を構成する殻タンパク質のアミノ酸の数（３４５残基）に極めて近い。このことから、表１の番号２〜３に示すタンパク質は、P.furiosusの殻タンパク質と同じ機能・構造をもつタンパク質であることが推測され、この発明者の解析したウイルス様粒子が、Sulfolobus属にも存在する可能性もあるといえる。
【０１２９】
表１の番号４〜５に示すタンパク質の種であるPyrococcus abyssiとPyrococcus horikoshiiは、P.furiosusと同じPyrococcus属で、菌の生育条件も似ており、各々のタンパク質の類似性も非常に高い。この類似検索で見つかったタンパク質は、どちらも１０１残基であり、発明者の発見したウイルス様粒子を構成するタンパク質の３４５残基と異なっている。しかし、Ｎ末から前半約１００残基の部分に限り、類似性が５０％を超えているという結果であった。図２１には、P.furiosusの殻タンパク質のアミノ酸配列と、Pyrococcus horikoshii PH1734のアミノ酸配列との相同性を示しており、５３％の類似性があることが分かる。Pyrococcus属で後半約２５０残基の類似性の高い部分が存在しないことは、進化の過程上で何らかの原因で淘汰され、失われた可能性があるといえよう。
【０１３０】
また、解析したウイルス様粒子の立体構造の結果とこの相同性検索の結果とから、ウイルス様粒子を構成する殻タンパク質の前半部分と後半部分とが、まるで異なるタンパク質であるかのように、機能・構造が分離されていると考えられる。このように考えられるのは、次の理由による。
【０１３１】
（理由１：構造上の理由）ウイルス様粒子のモノマー構造において、ウイルス様粒子の殻部を構成している部分は、後半の約２５０残基だけであり、前半のＮ末から１００残基ほどは、粒子の内部にありタンパク質構造がゆらいでいると考えられる。そのため、前半の１００残基は電子密度図で確認できていない。図２２は、ウイルス様粒子のモノマー構造を示しており、後半の２５０残基のリボン図で前半の１００残基は確認できないことを示している。
【０１３２】
（理由２：相同性検索結果による理由）相同性の検索において、Sulfolobus属のタンパク質のアミノ酸配列との比較を注意深く見ると、前半１００残基の配列の類似性が３７％で、後半２５０残基の配列の類似性は２０％であり、前半と後半との相同性が異なっている。さらに、P.abysii及びP.horikoshiiは、表１に記載したように、前半１００残基の配列の類似性が５０％を超えているけれども、後半２５０残基の類似配列はない。つまり、類似性からも、前半と後半とが分離されている。
【０１３３】
よって、後半の２５０残基部分が、ウイルス様粒子の殻部を構成している部分であって、前半の１００残基部分は、ウイルス内部で核酸などを認識している機能部分である可能性が強い。このことから、同じPyrococcus属でも、P.furiosus由来のタンパク質だけがウイルス様粒子となり、前半１００残基部分のタンパク質しかないP.abysii及びP.horikoshii由来のタンパク質では粒子とならないことが考えられる。
（実施例６ 大腸菌を用いた発現系）
この熱安定のウイルス様粒子の応用として、大腸菌を用いた発現系を構築した。まず、発現ベクターとしてpET9及びpET11を、ホストとしてBL211(DE3)pLysS及びBL21(DE3)Codon+を用いて、ウイルス様粒子を構成するタンパク質を発現させた。次に、ＳＤＳ−ＰＡＧＥにより、目的タンパク質の発現を確認した。さらに、タンパク質を遠心分離により精製した。その精製は、まず、超音波により細胞を破砕後、加熱処理（８０℃、１時間）を行った。次に、Sephadex200によるゲルろ過後、スクロース不連続密度勾配遠心分離（４０−１０％、２００００ｒｐｍ、１０時間）により、タンパク質を精製した。そして、タンパク質の精製後、電子顕微鏡により、球殻構造の形成を確認した。
【０１３４】
なお、大腸菌を用いた発現系の条件下でも、球殻構造が形成していることを確認している。その条件とは、Ｔｒｉｓ-ＨＣｌ(pH7.5)（２０ｍＭ）、グリセロール（１０％）、ＥＤＴＡ（５ｍＭ）、ＮａＣｌ（１００ｍＭ）である。
【０１３５】
このように、大腸菌を用いて殻タンパク質を発現することと、球殻構造が形成することとを確認した。これは、本発明の遺伝子及びタンパク質を用いれば、ウイルス様粒子（球状粒子）を大量に生産できるという可能性を示すものである。
【０１３６】
上記実施例１〜６により、次のことが言える。
（１）ウイルス様粒子は、直径３０ｎｍである。
（２）ウイルス様粒子は、超好熱菌P.furiosusの中に共生しながら存在している。
（３）ウイルス様粒子の殻タンパク質は、２つの機能、構造をもっている。
（４）大腸菌を用いた発現系により、球状構造を形成させることができる。
【０１３７】
また、相同性検索から、Sulfolobus属の２種で同様なウイルス様粒子が存在する可能性があり、同じPyrococcus属の中においては、P.furiosus由来のものだけがウイルス様粒子となることが予想される。そのため、ウイルス様粒子は、進化の上でも興味深い対象であることが分かった。
【０１３８】
【発明の効果】
本発明の遺伝子は、以上のように、球状粒子を構成するタンパク質をコードしている。それゆえ、この遺伝子により、球状粒子を構成するタンパク質を容易に合成して、さらに、そのタンパク質を利用して、ウイルス様粒子（球状粒子）を容易に製造することができる、という効果を奏する。
【０１３９】
また、本発明のタンパク質は、以上のように、集合することによって球状粒子を形成するタンパク質である。さらに、そのタンパク質をコードしている遺伝子は特定されている。それゆえ、球状粒子（ウイルス様粒子）の製造及び利用が容易になるという効果を奏する。
【０１４０】
また、本発明の球状粒子は、上記タンパク質を有することを特徴としている。それゆえ、上記タンパク質を集合させるといった方法により、容易に球状粒子を製造して利用することができるという効果を奏する。
【０１４１】
【配列表】

【図面の簡単な説明】
【図１】構造解析により得られた、ウイルス様粒子（球状粒子）の構造を示す模式図及び断面図である。
【図２】本発明の実施例の疎水性クロマトグラフィーにおける、溶出分画番号と吸光度との関係を示すグラフである。
【図３】本発明の実施例の疎水性クロマトグラフィーで得られた溶出分画液を、ＳＤＳ−ＰＡＧＥにかけたときの結果を示す模式図である。
【図４】結晶化前のサンプル、カラムクロマトグラフィーで分けたサンプル、及び結晶溶解液のサンプルの３つを、ＳＤＳ−ＰＡＧＥにかけたときの結果を示す模式図である。
【図５】結晶化前のサンプルの観察結果を示す電子顕微鏡写真である。
【図６】疎水性カラムクロマトグラフィーで分けたサンプルの観察結果を示す電子顕微鏡写真である。
【図７】結晶溶解液のサンプルの観察結果を示す電子顕微鏡写真である。
【図８】細菌ウイルスの形態を説明する説明図である。
【図９】部分アミノ酸配列の解析の結果、５残基目のＰ（プロリン）が見つかったときの、Ｎ末シークエンス法のＨＰＬＣの結果を示す模式図である。
【図１０】ＳＤＳ−ＰＡＧＥを行った後の目的タンパク質を、質量分析計で解析した結果を示す模式図である。
【図１１】 P.furiosusデータベースで見つかったタンパク質（ＰＦ１１９１及びＰＦ１１９２）のアミノ酸配列を示す模式図である。
【図１２】タンパク質合成における可能な３通りの読み枠を示す模式図である。
【図１３】本発明の実施例において、データベースのデータ及び解析結果をもとに、目的部分の塩基配列と対応するアミノ酸配列とを示した模式図である。
【図１４】図１３の続きを示す模式図である。
【図１５】 P.furiosusのゲノムＤＮＡからＰＣＲで目的ＤＮＡを増幅させ、目的アミノ酸配列を読むことを示す模式図である。
【図１６】データベース上にあるP.furiosusの塩基配列をもとに、得られるＤＮＡがフレームシフト部分を含む約２４０塩基になるように作製したプライマーと、P.furiosusのゲノムＤＮＡとを用いてＰＣＲを行い、そのＰＣＲ産物を電気泳動にかけたときの結果を示す模式図である。
【図１７】解析により得られたＤＮＡの塩基配列と、データベースにある塩基配列とを一塩基ずつ比較したときの結果を示す模式図である。
【図１８】修正した塩基配列と対応するアミノ酸配列の一部とを併記して、２つのタンパク質（PF1191及びPF1192）がつながることを説明する説明図である。
【図１９】修正した１０３８塩基の全塩基配列と、それに対応する３４５残基の全アミノ酸配列とを示す図である。
【図２０】 Sulfolobus tokodaii ST 0992のアミノ酸配列とP.furiosusのアミノ酸配列との相同性を示す模式図である。
【図２１】 P.furiosusの殻タンパク質のアミノ酸配列と、Pyrococcus horikoshii PH1734のアミノ酸配列との相同性を示す模式図である。
【図２２】ウイルス様粒子のモノマー構造（ウイルス様粒子の殻タンパク質の構造）を示す立体構造図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein constituting a spherical particle (virus-like particle) and a novel gene encoding the protein.
[0002]
[Prior art]
There are roughly three living worlds on the earth: Eukarya (Eukaryotes), Bacteria (Archaea) and Archea (Archaea), and mammals including humans are at the apex of eukaryotes.
[0003]
Although archaea were classified as eubacteria until about 20 years ago due to their similar appearance, as a result of structural analysis of genes, they were recognized as organisms that are different from eubacteria, and are intermediate between eubacteria and eukaryotes. Was positioned. It is characterized by being in a special environment and grows at very high temperatures and very high salt concentrations. The conditions that make it easy for us to live are those that have adequate temperature, air, sunlight, water, food, etc., but those that do not have such appropriate temperature, air, sunlight, water, food, etc. This is the condition for the growth of bacteria.
[0004]
Archaea are divided into subdomains of Euryarchaeota close to eukaryotes and Crenarchaeota close to eubacteria. Archaeal Pyrococcus is a hyperthermophilic bacterium classified as Euryarchaeota. Pyrococcus furiosus (hereinafter referred to as “P.furiosus” where appropriate) was found in the extreme environment of a sulfurous volcano. It is an anaerobic bacterium that grows in a state of low oxygen, and is a hyperthermophilic bacterium that grows at a high temperature of 70 ° C. to 103 ° C. and a high pH (see Non-Patent Document 1).
[0005]
Many researches have been made on the hyperthermophilic bacterium P. furiosus with this unique heat-resistant mechanism as a research target. Factors of the heat-resistant mechanism understood to date are (1) the fact that DNA (deoxyribonucleic acid) is hardly destroyed in the extreme environment and the chromosome is completely maintained, and (2) the DNA repair enzyme is involved. It is said that the DNA repair system of the system is capable (see Non-Patent Document 2).
[0006]
Also, as an application, since hyperthermophilic bacteria are heat-resistant, the protein is also heat-stable, hardly denatured and easily crystallized, and is said to be advantageous for protein experiments. In addition, DNA polymerase derived from the hyperthermophilic bacterium P. furiosus is widely used industrially, such as being mass-produced for use in the Polymerase chain reaction (PCR) method (see Non-Patent Document 3). .
[0007]
By the way, thanks to the rapid development of biological science in recent years, proteins with important functions related to enzyme reactions, molecular recognition processes and catalytic processes essential for life activities can be analyzed at the atomic resolution by X-ray crystal structure analysis. It is possible to study.
[0008]
The inventor of the present application has been studying the elucidation of the protein synthesis mechanism by the supramolecular complex ribosome, and has also performed an atomic structure analysis of the 70S ribosome derived from the hyperthermophilic bacterium P. furiosus, which is important in the research. . Then, 70S ribosome of high purity was extracted from the hyperthermophilic bacterium P. furiosus, and further a crystal thought to be 70S ribosome was obtained, and X-ray crystal structure analysis of the crystal was performed. The crystallization conditions at that time are shown below.
(Crystallization conditions)
Method Hanging drop vapor diffusion method
Buffer 20 mM Tris-HCl
Protein 40-60mg / ml
Precipitant MPD
Precipitant concentration in crystallization drop 0.5% (w / v)
Precipitant concentration in the crystallization reservoir 16% (w / v)
Temperature 25 ° C
However, during the analysis, the presence of a shell structure not found in the 70S ribosome was confirmed in the crystal structure, and the crystal structure was found to be a virus-like particle having a shell structure. This indicates that the crystals were not 70S ribosome crystals but virus-like particle crystals.
[0009]
The above results were completely unexpected for the inventors. The 70S ribosome sample for crystallization was from the 70S ribosome as a result of SDS-PAGE and activity measurement, but it was virus-like particles that were crystallized. In addition, “SDS” of SDS-PAGE means sodium dodecyl sulfate (dodecyl sulfate, sodium salt). In addition, “PAGE” in SDS-PAGE is polyacrylamide gel electrophoresis.
[0010]
As described above, 70S ribosome crystals were not obtained. However, this unexpected virus-like particle derived from hyperthermophilic bacteria has an interesting point as a research object. One is that there are no reports of Euryarchaeota virus close to eukaryotes, and of course, its structure and function are not known at all (see Non-Patent Document 4). In addition, the inventors proceeded with the structural analysis of this virus-like particle and determined the structure by the heavy atom isomorphous substitution method. As a result, the above virus-like particle is a spherical particle with a diameter of about 30 nm having 180 = shell protein and T = 3 icosahedral symmetry, and about 250 residues from the C-terminal of the shell protein are virus-like particles. I confirmed that I was involved in the skeleton. Moreover, it has confirmed that the whole structure was similar to the structure of Capsid of bacteriophage HK97 (refer nonpatent literature 5).
[0011]
[Non-Patent Document 1]
J Biol Chem 264 (9) 5070-9 (1989)
[0012]
[Non-Patent Document 2]
J Bacteriol 177 (21) 6316-8 (1995)
[0013]
[Non-Patent Document 3]
Gene 108 (1) 1-6 (1991)
[0014]
[Non-Patent Document 4]
FEMS Microbiology Reviews 18 225-236 (1996)
[0015]
[Non-Patent Document 5]
Science 289 2129-2133 (2000)
[0016]
[Problems to be solved by the invention]
However, with regard to the above virus-like particles, for example, the biochemical properties of virus-like particles are hardly known other than the above, and it is extremely difficult to use virus-like particles (spherical particles). There is a point. The virus-like particles can be used, for example, for drug delivery systems (DDS) by packing drugs, genes, etc. inside virus-like particles having a heat-stable structure. Can be mentioned.
[0017]
The present invention has been made in view of the above conventional problems, and its purpose is to elucidate the biochemical properties of virus-like particles (spherical particles) and to facilitate the use of the spherical particles. It is to provide a gene and a protein necessary for the preparation, and to provide spherical particles obtained by using these genes and the like.
[0018]
[Means for Solving the Problems]
In view of the above problems, the present inventor has intensively studied the virus-like particles. First of all, we clarified why virus-like particles existed in hyperthermophilic bacteria, and investigated what kind of function virus-like particles have. Then, it was examined whether or not virus-like particles were present in the 70S ribosome sample for crystallization, and further, whether or not nucleic acids were contained inside the virus-like particles was examined. Furthermore, the analysis of the amino acid sequence of the protein considered to be the shell protein of the virus-like particle and the analysis of the DNA base sequence were performed, and the similarity search of various species was examined. As a result, the present inventor has determined the amino acid sequence of the shell protein and the base sequence of the gene encoding the protein, and has completed the present invention.
[0019]
The gene of the present invention encodes any one of the following proteins (a) to (c).
(A) a protein having the amino acid sequence of SEQ ID NO: 3;
(B) The amino acid sequence shown in SEQ ID NO: 3 has an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and aggregates to form a spherical particle protein.
(C) A protein having the amino acid sequence set forth in SEQ ID NO: 6 and forming a spherical particle by assembling.
[0020]
The protein (a) is a shell protein that constitutes spherical particles (virus-like particles) derived from P. furiosus. The spherical particles (virus-like particles) are particles derived from the hyperthermophilic bacterium P. furiosus and are spherical shell structures (PfV) having regular icosahedral symmetry. The spherical particles have a diameter of 30.5 nm to 36.3 nm (305 to 363 angstroms) as shown in FIG. The spherical particles are particles obtained by associating 180 single polypeptide chains (the protein of (a) above) with non-covalent bonds. And this spherical particle exists stably also in high temperature conditions, such as 90 to 100 degreeC so that the origin called hyperthermophilic P.furiosus may show. Further, the spherical particles have a function of allowing molecules present in the particles, such as RNA (ribonucleic acid), to exist stably even under the high temperature conditions.
[0021]
As described above, the spherical particles are stable under high-temperature conditions, and have a function of allowing molecules present in the particles to exist stably. Therefore, for example, by packing drugs, genes, and the like inside the spherical particles, use of the spherical particles in a drug delivery system (Drug Delivery System: DDS) can be expected.
[0022]
The gene of the present invention is a novel gene encoding the shell protein. As a result, a gene for facilitating the use of spherical particles (virus-like particles) can be provided.
[0023]
In addition, examples of the gene according to the present invention include the following genes (A) to (F). Use of these genes facilitates the use of spherical particles (virus-like particles).
(A) A gene having the base sequence set forth in SEQ ID NO: 1.
(B) Of the base sequence described in SEQ ID NO: 1, one or several bases have a base sequence substituted, deleted, inserted, and / or added, and form a spherical particle by assembling. A gene characterized by encoding a protein.
(C) The gene according to (B), wherein the protein has the amino acid sequence described in SEQ ID NO: 3.
(D) The protein has an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 3. The gene according to (B).
(E) A gene having the base sequence set forth in SEQ ID NO: 4 and encoding a protein that forms a spherical particle by assembling.
(F) Of the base sequence set forth in SEQ ID NO: 4, one or several bases have a base sequence substituted, deleted, inserted and / or added, and form spherical particles by assembling. A gene characterized by encoding a protein.
[0024]
Note that “one or several bases are substituted, deleted, inserted, and / or added” means that the number of bases that are possible by a known DNA recombination technique, a point mutation introduction method, etc. Means a substitution, deletion, insertion, and / or addition. In addition, “one or several amino acids are substituted, deleted, inserted, and / or added” is as many as possible by a known mutant protein production method such as site-directed mutagenesis. It means that (preferably 10 or less, more preferably 7 or less, more preferably 5 or less) amino acids have been substituted, deleted, inserted and / or added.
[0025]
By the way, “gene” of the present invention includes DNA and RNA. The DNA referred to herein includes, of course, DNA (for example, cDNA (complementary DNA), genomic DNA, etc.) obtained by cloning, chemical synthesis techniques, or a combination thereof. The DNA may be double-stranded or single-stranded, and the single-stranded DNA may be a coding DNA that serves as a sense strand or an anti-coding strand that serves as an antisense strand. This antisense strand can be used for probes and the like.
[0026]
Furthermore, the “gene” of the present invention includes sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences) in addition to the sequences encoding the proteins (a) to (c) above. It may be a thing.
[0027]
As described above, a sequence listing may be used in describing the gene of the present invention. In the sequence table, the base sequence of DNA is shown for convenience of explanation. However, when the gene of the present invention refers to RNA, the base “T (thymine)” shown in the sequence listing is interpreted as “U (uracil)”.
[0028]
The protein of the present invention is characterized by having the amino acid sequence set forth in SEQ ID NO: 3. This protein is a shell protein constituting spherical particles (virus-like particles) derived from P. furiosus. The amino acid sequence of this protein is the amino acid sequence determined by the present inventors for the first time. This will be described in detail in the examples shown below. As described above, the gene encoding the protein of the present invention is a novel gene identified by the present inventors.
[0029]
As a result, it is possible to provide a protein that elucidates the biochemical properties of the virus-like particles and facilitates the utilization of the virus-like particles (spherical particles).
[0030]
Furthermore, examples of the protein according to the present invention include the following (i) to (ii). (i) The amino acid sequence of SEQ ID NO: 3 has an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added, and aggregates to form a spherical particle Protein characterized by this.
(ii) A protein having the amino acid sequence set forth in SEQ ID NO: 6 and forming a spherical particle by assembling.
[0031]
The protein (ii) is a protein consisting of 245 amino acid residues excluding the first 100 residues of the amino acid sequence shown in SEQ ID NO: 3. This part consisting of 245 amino acid residues is the part constituting the shell of spherical particles. Therefore, a protein having a portion consisting of 245 amino acid residues is very likely to constitute a spherical particle. The above protein consisting of 245 (about 250) amino acid residues excluding the first 100 residues will be described in detail in Examples below.
[0032]
The protein according to the present invention mainly refers to a protein isolated and purified from cells or the like. However, the protein according to the present invention may be in a state where a gene encoding the protein is introduced into a host cell and the protein is expressed in the cell. Furthermore, the protein according to the present invention may contain an additional polypeptide. Examples of the protein containing an additional polypeptide include proteins labeled with epitopes such as HA, Myc, and flag.
[0033]
The spherical particles of the present invention are characterized by having the protein described above.
[0034]
As described above, the proteins constituting the spherical particles of the present invention aggregate to form spherical particles (spherical shell structure: PfV). Furthermore, the inventor has specified a gene encoding the protein. Therefore, spherical particles having heat resistance can be easily obtained.
[0035]
DETAILED DESCRIPTION OF THE INVENTION
An embodiment of the present invention will be described as follows. In addition, this invention is not limited to the following embodiment, A various change is possible within the scope of the present invention.
[0036]
(1) About the gene of this embodiment
The gene in this Embodiment is demonstrated. The gene of the present embodiment is a gene derived from the hyperthermophilic bacterium P. furiosus and encodes a protein constituting a spherical particle (virus-like particle).
[0037]
Examples of the base sequence of the gene in the present embodiment are shown in SEQ ID NOS: 1 and 4. The gene having the base sequence of SEQ ID NO: 1 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 3. A combination of the base sequence of SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 2.
[0038]
The gene having the base sequence of SEQ ID NO: 4 encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 6. A combination of the base sequence of SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 5.
[0039]
Next, a method for obtaining a gene in the present embodiment will be described. The method for obtaining the gene of the present embodiment is not particularly limited. For example, it is stored in the P.furiosus database (http://www.genome.utah.edu/sequence.html) and the nucleotide sequence data described in the sequence listing (SEQ ID NOs: 1-2 and 4-5). The DNA fragment containing the gene of this embodiment can be isolated and cloned by various methods based on the data of the nucleotide sequence present.
[0040]
Specifically, the gene of the present embodiment can be easily obtained by using amplification means such as PCR. First, the DNA sequence described in the sequence listing is compared with the data stored in the P. furiosus database, so that the region encoding the protein to be obtained is amplified. Primers are prepared from the sequence of the untranslated region (or its complementary sequence). Next, when PCR is performed using these primers and P. furiosus DNA (genomic DNA, cDNA, etc.) as a template, the DNA sandwiched between the two primers is amplified. Thereby, a large amount of DNA fragments containing the gene of the present embodiment can be obtained.
[0041]
In addition, as a method for obtaining a base sequence in which one or several bases are substituted, deleted, inserted, and / or added in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 4, for example, a site Specific mutagenesis (Hashimoto-Gotoh, Gene 152, 271-275 (1995), etc.), mutation by transposon insertion, PCR method and the like can be mentioned.
[0042]
(2) Proteins and spherical particles of this embodiment
The protein of the present embodiment is a shell protein constituting a spherical particle (virus-like particle). In addition, the spherical particle of the present embodiment is a particle formed by associating 180 single polypeptide chains (that is, the protein of the present embodiment) with a non-covalent bond.
[0043]
Next, the protein acquisition method in the present embodiment will be described. The method for obtaining a protein in the present embodiment is not particularly limited. For example, first, the gene of the present embodiment is incorporated into a host by a well-known method. In addition, as a host said here, microorganisms (E. coli, yeast, etc.), an animal cell, etc. are mentioned, for example. Next, the protein of the present embodiment can be easily obtained by expressing the incorporated gene and obtaining and purifying the protein encoded by the gene.
[0044]
In addition, when the gene of the present embodiment is incorporated into a host, a promoter sequence is appropriately selected according to the type of host cell in order to reliably express the gene, and this and the gene according to the present invention can be used for various plasmids, etc. What has been incorporated into may be used as an expression vector.
[0045]
Further, the method of introducing the expression vector into the host cell, that is, the transformation method is not particularly limited, and a conventionally known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method is preferably used. Can do.
[0046]
Various markers may be used to confirm whether or not the gene of the present embodiment has been introduced into the host cell, and whether or not the gene is surely expressed in the host cell. For example, a gene deleted in the host cell is used as a marker, and a plasmid or the like containing this marker and the gene of this embodiment is introduced into the host cell as an expression vector. Thus, the introduction of the gene of the present embodiment can be confirmed from the expression of the marker gene. Alternatively, the protein according to the present invention may be expressed as a fusion protein. For example, the green fluorescent protein GFP (Green Fluorescent Protein) derived from Aequorea jellyfish may be used as a marker, and the protein according to the present invention may be expressed as a GFP fusion protein. Good.
[0047]
As described above, when a foreign gene is introduced into a host, there are various expression vectors and hosts that incorporate a promoter that functions in the host in the recombination region of the foreign gene. Should be selected. The method for extracting the produced protein varies depending on the host used and the nature of the protein, but the target protein can be purified relatively easily by using, for example, a tag.
[0048]
Further, the method for producing the mutant protein is not particularly limited. For example, a site-directed mutagenesis method (Hashimoto-Gotoh, Gene 152, 271-275 (1995), etc.), a PCR method or the like may be used to introduce a point mutation into a base sequence to produce a mutant protein. Alternatively, a well-known mutant protein production method such as a mutant strain production method by inserting a transposon may be used. Thus, a mutant protein can be easily produced by modifying the base sequence of the gene obtained by the above-described method so that one or more bases are substituted, deleted, inserted, and / or added. can do. Furthermore, a commercially available kit (for example, QuikChange Site-Directed Mutagenesis Kit manufactured by Stratagene) may be used for the production of the mutant protein.
[0049]
Furthermore, an antibody can be prepared using the protein of the present invention or a partial peptide thereof as an antigen. The method for producing the antibody is not particularly limited, and a polyclonal antibody or a monoclonal antibody may be produced by a known method. Known methods include, for example, the literature (Harlow et al., “Antibodies: A laboratory manual (Cold Spring Harbor Laboratory, New York (1988)), Iwasaki et al.,“ Monoclonal antibody hybridoma and ELISA, Kodansha (1991) ””. The antibody prepared in this way is effective for detecting the protein of the present invention.
[0050]
(3) About the spherical particles of the present embodiment
The spherical particles of the present embodiment are particles derived from the hyperthermophilic bacterium P. furiosus and are composed of the above protein.
[0051]
Next, a method for obtaining spherical particles in the present embodiment will be described. As will be described in detail in the following Examples, spherical particles can be obtained by culturing P. furiosus, disrupting the cells, and centrifuging and hydrophobic chromatography.
[0052]
The acquisition method is a method by culturing P. furiosus. However, spherical particles can also be obtained by synthesizing proteins using the genes of this embodiment and assembling the synthesized proteins. For example, if the protein of this embodiment is synthesized by a large-scale expression system using Escherichia coli or the like, a higher-order structure can be easily formed by self-association.
[0053]
(4) Usefulness
The gene, protein, and spherical particle of the present embodiment are particles derived from the hyperthermophilic bacterium P. furiosus. Moreover, the spherical particles of the present embodiment can be manufactured by assembling 180 proteins (protein particles) of the present embodiment.
[0054]
As can be seen from the fact that it is derived from the hyperthermophilic bacterium P. furiosus, the spherical particles (spherical shell structure: PfV) of the present embodiment exist stably even under high temperature conditions such as 90 ° C. to 100 ° C. Further, the spherical particles have a function of allowing molecules existing in the particles, such as RNA, to stably exist even under the high temperature conditions.
[0055]
For example, the spherical particles of the present embodiment can be used as a base material for DDS (drug delivery system) by packing drugs, genes, and the like inside the spherical particles. Further, the use of the spherical particles in the present embodiment includes, for example, use as a heat-resistant capsule that stably stores the material packed inside the spherical particles under high temperature conditions, use as a cage compound, and the like. . And if the function which protects the molecule | numerator which exists in the particle | grains of a spherical particle from a heat | fever is utilized, the application to the protector of the molecule | numerator of a nano level magnitude | size is also considered.
[0056]
In the use for the DDS or the storage capsule, the spherical particles are constituted by non-covalent bonds of a plurality of proteins, which can be a factor for increasing the utility value of the spherical particles. For example, it is considered that spherical particles can be easily dissociated under appropriate conditions (for example, conditions where the salt concentration is increased).
[0057]
Further, based on the three-dimensional structure of the spherical particles in the present embodiment, for example, it is easy to confine the target molecule inside the spherical particle by specifically binding the molecule to an arbitrary region of the polypeptide chain. It is believed that there is. Furthermore, by appropriately modifying the outside of the spherical particles, the properties of the entire particles can be easily controlled. The fact that the role difference between the first half 100 residues and the second half about 250 residues in the amino acid sequence shown in SEQ ID NO: 3 is known is that the use of spherical particles is considered based on the three-dimensional structure as described above. This is valuable information. In addition, since the addition of an epitope or a specific binding function is also possible, it is thought that the spherical particle of this Embodiment can be utilized for various uses.
[0058]
Furthermore, since the gene encoding the protein of the present embodiment has been specified, it is easy to alter or modify the protein constituting the spherical particle. That is, it is also easy to use the modified or modified protein. Furthermore, it is easy to control the function in a three-dimensional structure-specific manner by mutating or modifying amino acids at specific sites.
[0059]
Unlike similar structures using conventional synthetic polymers, spherical particles using polypeptide chains have specific interactions between subunits, formed spherical shell structures, or between target molecules. It is high and its utility value is considered high.
[0060]
【Example】
Examples of the present invention are shown below. In addition, this invention is not limited to the following Example, A various change is possible within the scope of the present invention.
[0061]
(Example 1 Separation and purification of virus-like particles)
First, in order to verify whether the virus-like particles found in the crystal structure exist in the ribosome sample before crystallization, the above sample was further purified to try to separate the ribosome and virus-like particles. .
[0062]
A sample for crystallization of P. furiosus-derived 70S ribosome was obtained and purified as follows. First, P. furiosus was cultured. P. furiosus is cultured at 98 ° C. D. Bacteria were collected when (550 nm) was about 0.7. Thereafter, the cells were washed with a washing solution.
[0063]
Next, the cells were crushed with a mortar containing quartz sand, and centrifuged (8000 rpm, 20 minutes) to obtain a supernatant. The supernatant was further centrifuged (18000 rpm, 1 hour) to obtain a supernatant again. Next, the supernatant was centrifuged (40000 rpm, 3 hours) to obtain a precipitate.
[0064]
Next, the precipitate was dissolved in a potassium chloride buffer, and the lysate was subjected to sucrose discontinuous density gradient centrifugation (1.6 M sucrose, 40000 rpm, 20 hours). The precipitate obtained by the sucrose discontinuous density gradient centrifugation was dissolved again in the buffer and then reacted with puromycin buffer. After the reaction, a sample for crystallization of 70S ribosome was obtained by obtaining a precipitate by glycerol discontinuous density gradient centrifugation (25% (v / v) glycerol, 40000 rpm, 15 hours). By the above operation, about 300 mg of a sample for crystallization of 70S ribosome was obtained from 96 liters of medium.
[0065]
Next, in order to examine whether or not virus-like particles were mixed in the sample for crystallization of 70S ribosome obtained by the above operation, the sample for crystallization was further subjected to hydrophobic chromatography. This hydrophobic chromatography utilizes the fact that the hydrophobic bond of a protein becomes strong under high ionic strength. Specifically, the protein is first adsorbed onto the column in the presence of concentrated ammonium sulfate. Then, the protein is eluted from the column using an elution gradient prepared so that the salt concentration decreases.
[0066]
Next, the hydrophobic chromatography method will be described. First, the ammonium sulfate concentration was added to the final concentration of 1.7 M to the ribosome crystallization sample obtained by the above method. Next, the denatured product was removed by centrifugation (1000 rpm, 10 minutes).
[0067]
Next, the carrier TSKgel Butyl-Toyopearl 650 (manufactured by Tosoh Corporation) was equilibrated with 300 ml of equilibration buffer twice the column volume, and then the protein solution was adsorbed. Then, after washing with 50 ml equilibration buffer, elution was performed by reverse phase chromatography with a linear concentration gradient (ammonium sulfate concentration 1.5 M to 0 M) of 150 ml elution buffer and 150 ml equilibration buffer. The obtained eluate was applied to a fraction collector and subjected to SDS-PAGE. The fraction size was 5 ml per bottle.
[0068]
The composition of the equilibration buffer was Tris-HCl pH 7.5 (20 mM), ammonium chloride (75 mM), magnesium chloride (30 mM), potassium chloride (0.2 M), EDTA (ethylenediamine-N, N, N ′, N′-tetraacetic acid) (0.25 mM), mercaptoethanol (5 mM), and ammonium sulfate (1.5 M). The composition of the elution buffer was Tris-HCl pH 7.5 (20 mM), ammonium chloride (75 mM), magnesium chloride (30 mM), potassium chloride (0.2 M), EDTA (0.25 mM), mercaptoethanol (5 mM) did. Note that “M” which is a unit of concentration indicates mol / l. The above “Tris” means (tris (hydroxymethyl) aminomethane).
[0069]
A graph showing the relationship between the elution fraction number and the absorbance in the hydrophobic chromatography is shown in FIG. In addition, FIG. 3 shows the results when the eluted fraction was subjected to SDS-PAGE.
[0070]
As shown in FIG. 3, when the elution fraction was subjected to SDS-PAGE, a 40 kDa main band considered to be a coat protein of virus-like particles appeared first (fraction Nos. 54 to 62), and later ribosomal ribosomes. A number of bands (fraction numbers 70-78) that are considered proteins are followed. This indicates that ribosome and virus-like particles were separated by hydrophobic column chromatography. Therefore, the samples of fraction numbers 54 to 62 considered to be virus-like particles were concentrated and used as samples separated by columns.
[0071]
(Example 2 Form of virus-like particles)
In order to confirm that the 40 kDa sample separated by hydrophobic column chromatography in Example 1 was the same virus-like particle as the crystal, the following experiment was conducted. First, a sample before crystallization, a sample separated by column chromatography, and a sample of a crystal solution were prepared, and confirmed by SDS-PAGE and an electron microscope. The crystal solution is a solution in which crystals precipitated by the hanging drop vapor diffusion method are collected in an Eppendorf tube, crushed with a glass rod, and dissolved in a buffer from which the precipitant has been removed.
[0072]
The results of confirming the above three samples by SDS-PAGE are shown in FIG. In FIG. 4, lane 1 shows the molecular weight marker, lane 2 shows the sample before crystallization, lane 3 shows the sample separated by hydrophobic column chromatography, and lane 4 shows the result of the crystal solution.
[0073]
According to FIG. 4, a large number of ribosomal protein bands can be confirmed in the sample before crystallization (lane 2). In the crystal lysate (lane 4), a 40 kDa band that appears to be a shell protein of virus-like particles appears, as in the sample separated by column chromatography (lane 3).
[0074]
Moreover, the electron microscope observation of the three samples was performed as follows. First, three samples were placed on a microgrid for an electron microscope for 10 seconds, and an excessive amount of sample was absorbed with paper. Next, a 2% (w / v) uranyl acetate solution for negative staining was placed on the grid for 1 minute. This uranyl acetate solution is added because a fine structure is clearly dyed as a result of heavy metal atoms having a strong electron scattering ability covering the surface of the sample and penetrating into the fine gaps. An excess amount of the solution was absorbed with paper and air-dried, and the grid was attached to an electron microscope and observed.
[0075]
The observation result by this electron microscope is shown in FIGS. 5 shows the observation result of the sample before crystallization, FIG. 6 shows the observation result of the sample separated by hydrophobic column chromatography, and FIG. 7 shows the observation result of the sample of the crystal solution. As shown in FIG. 5, many ribosome particles could be observed in the sample before crystallization. And some virus-like particles could be observed. On the other hand, as shown in FIGS. 6 and 7, in the microscopic observation of the sample separated by column chromatography and the sample of the crystal lysate, many particles (virus-like particles) having a diameter of about 30 nm could be observed.
[0076]
The above results indicate that when the sample before crystallization obtained by purifying 70S ribosome from P. furiosus culture is further subjected to hydrophobic chromatography, only virus-like particles can be purified without crystallization.
[0077]
From these experimental results, it can be seen that a small amount of virus-like particles are mixed in a large amount of ribosome particles in the sample before crystallization, and the small amount of virus-like particles precipitated as crystals when crystallized. I understand. In the first place, it is said as a well-known fact that protein crystallization does not crystallize unless the protein is present in high purity, but it can be said that completely different results were obtained in this example.
[0078]
Similar to this example, crystals were deposited aiming to crystallize the ribosome, but it was confirmed before that other proteins were crystallized instead of the ribosome itself. As a background to this accidental crystallization, the ribosome has a high hydrophilicity. The ribosome is thought to play a role in reducing the solubility of proteins such as PEG (polyethylene glycol), which is a precipitant used for crystallization.
[0079]
Moreover, as a form of virus-like particle | grains, it turned out that the diameter of particle | grains is about 30 nm from the result of X-ray structural analysis. As a result of viewing the virus-like particles with an electron microscope, it was confirmed that the particle diameters were consistent.
[0080]
Currently, existing viruses can be classified into three categories: bacterial viruses (bacteriophages), animal viruses, and plant viruses. Assuming that this virus-like particle is a mature virus infected with P. furiosus as a host, it can first be classified as a bacterial virus.
[0081]
There are two types of bacterial viruses: one with a head and tail and one without a tail. This virus-like particle does not have a tail as seen with an electron microscope. Represents type E (Levivirus). FIG. 8 shows the form of the bacterial virus. As shown in FIG. 8, bacterial virus forms can be classified into types A to F, and type E has RNA.
[0082]
However, other E-type bacteriophages that are currently identified have a diameter of 25 nm or less and all have a shell protein molecular weight of about 14,000 (Bacteriological reviews 31 230-311 (1967)). Since the molecular weight of the shell protein of the virus-like particle is as large as 40 kDa, it does not fall under this classification, and it can be said that there is no virus that is particularly similar to the virus-like particle that was found. However, the bacteriophage HK97 used as a model molecule after structural analysis by the inventor is a DNA virus having a tail (type A), and is a particle having a large head part with a diameter of 60 nm. However, bacteriophage HK97 and virus-like particles are similar in the structure of one shell protein and the characteristic structure in which the outer shell of the particle is thin.
[0083]
From the above, the virus-like particle discovered by the inventor may not be classified as a known virus even in terms of morphology, and may be the first virus of Euryarchaeota close to eukaryotes. For this reason, since detailed biochemical elucidation is necessary, the amino acid sequence of the shell protein constituting the virus-like particle was first determined, and the homology with other species was compared.
[0084]
(Example 3 Analysis of amino acid sequence of shell protein)
In order to verify the biochemical properties of the virus-like particle, the amino acid sequence of the shell protein constituting the virus-like particle was analyzed, and further the DNA base sequence was analyzed.
[0085]
4-1 Determination of partial sequence in amino acid sequence
What is necessary for analyzing the physiological function of a 40 kDa protein considered to be a shell protein of virus-like particles is structural information. By determining the entire amino acid sequence and the entire base sequence of the target protein, physiological functions can be roughly estimated based on homology with other proteins. Also in the three-dimensional structure analysis of proteins, the amino acid sequence information is indispensable information because the amino acid side chain structure of the target protein is fitted to the obtained electron density one by one.
[0086]
In order to actually determine the amino acid sequence, a standard method is to first determine a partial amino acid sequence, clone cDNA based on the information, and deduct the entire amino acid sequence from the base sequence. According to this method, the inventor first performed an N-terminal sequencing method using a protein sequencer and a mass spectrum method using a mass analyzer in order to determine a partial amino acid sequence.
[0087]
Here, the N-terminal sequencing method using a protein sequencer will be briefly described. The principle of the protein sequencer utilizes Edman degradation in which PITC (phenyl isothiocyanate) is coupled to the N-terminal amino group of the protein and the N-terminal amino acid is cleaved with trifluoroacetic acid. And it isolate | separates and identifies sequentially by a high performance liquid chromatography (HPLC), The amino acid sequence (10-20 residues) from N terminal is determined by repeating this reaction.
[0088]
The operation of the N-terminal sequencing method using a protein sequencer will now be described. After SDS-PAGE of the crystal solution, the gel and the pretreated PVDF membrane were sandwiched between filter papers and attached to a semi-blotting apparatus. Next, constant current (0.5 mA / cm ² ) For 60 minutes to transfer the protein to the PVDF membrane. Next, the transferred PVDF membrane was stained with a CBB staining solution, set in a protein sequencer, and analyzed. The “PVDF” of the PVDF membrane is polyvinylidin fluoride.
[0089]
As a result of the analysis of the partial amino acid sequence, it was possible to identify the N-terminal partial amino acid sequence of 12 residues out of 15 called MLKIXPXLIXYDKPL (X cannot be analyzed). FIG. 9 shows the results of HPLC by N-terminal sequencing when P (proline) at the fifth residue (the sixth residue when X is included) is found.
[0090]
Next, determination of a partial sequence by mass spectrometry will be described. Specifically, a band of about 40 KDa separated by SDS-PAGE was analyzed by mass spectrometry.
[0091]
Mass spectrometry that measures the exact molecular weight of proteins and their peptides can also be used for analysis of amino acid sequences. Mass spectrometer, Voyger ^TM Elite XL (PerSeptive Biosystems) was used. As an ion source for ionizing a sample, a MALDI method was adopted in which ionization is performed two-dimensionally by energy from matrix molecules excited by laser irradiation. A time-of-flight type called TOFMS was used for mass spectrometry. By using this TOFMS, metastable ions are read and amino acid sequences can be analyzed.
[0092]
In the operation, first, the gel of the target protein portion after performing SDS-PAGE was cut out with a scalpel, washed, and then subjected to reductive alkylation to cleave the disulfide bond. Then, enzymatic digestion with lysyl endopeptidase was performed, and the digested peptide was extracted from the gel portion using trifluoroacetic acid and acetonitrile solution, and desalted. The obtained digested peptide sample was mixed with a solution of matrix molecules, set in a mass spectrometer, and analyzed. The analysis result is shown in FIG.
[0093]
The results of amino acid sequence analysis obtained by the above analysis were searched in amino acid databases of various proteins, and further detailed investigations were made with the analysis values. As a result, characteristic partial peptides were analyzed and matched with the two proteins PF1191 and PF1192 in the P.furiosus database (http://www.genome.utah.edu/sequence.html) (see FIG. 11).
[0094]
FIG. 11 shows 96 amino acid residues of PF1192 and 261 amino acid residues of PF1191. And the bold and italic type (indicated as “bold” in the figure) and the underlined portion are both the results obtained by the N-end sequence method and the results obtained by the mass spectrum method, The part where the amino acid residue in P.furiosus database matched is shown. Moreover, the location shown in bold indicates the location where the result obtained by the mass spectrum method and the amino acid residue in the P. furiosus database match.
[0095]
The N terminal sequence (15 amino acid residues) of PF1192 in the database is “MLISPNTPLINRKPY” as shown in FIG. On the other hand, the result obtained by the N-terminal sequencing method is “MLKIXPXLIXYDKPL” as described above. When these are compared, a total of 9 residues of 1st to 2nd, 4th, 6th, 8th to 9th, and 12th to 14th match. And for the 6 mismatched residues (3rd, 5th, 7th, 10th to 11th and 15th), the results obtained by the N-terminal sequencing method are somewhat ambiguous in interpretation. It was. Therefore, when it examined again considering the result of HPLC, it turned out that the result of the N-terminal sequencing method is data consistent with the result of HPLC. As a result, it was concluded that the results by the N-terminal sequence method and the results by the mass spectrum method were in agreement. From this, it was concluded that the shell protein of the virus-like particle was P. furiosus PF1191 and PF1192 protein.
[0096]
This experimental result initially seemed to be an analytical error. This is because, even though the band portion of about 40 kDa was extracted, it matched with the two proteins.
[0097]
However, for the reasons (1) and (2) shown below, it could not be said that the result was merely an incorrect result.
(1) Add about 12 kDa and 28 kDa, which are the expected molecular weights of the two 96-residue and 261-residue proteins, to obtain the target of about 40 kDa.
(2) Two proteins found in a huge number of databases were immediately adjacent to each other in the DNA sequence.
[0098]
Presuming from the two points (1) and (2) above, a translation error occurs in the translation process of RNA expressed by the protein, and one base of RNA in the middle of the two proteins is read out. The hypothesis that protein was born was considered. Therefore, further experiments were continued.
[0099]
(Example 4 Determination of DNA base sequence and amino acid sequence)
There are three processes for protein expression: (I) DNA replication, (II) transcription into RNA, and (III) translation from RNA to protein. In the translation process, three base sequences of RNA are read in order, and a triplet of this base corresponds to one amino acid, and a protein is synthesized. Therefore, the amino acid sequence of the protein is determined by the base sequence of the gene. Furthermore, if the base sequence of the gene can be determined, the amino acid sequence is determined.
[0100]
As described above, the amino acid sequence is determined if the base sequence of the gene can be determined. However, in principle, RNA sequences can be translated in three reading frames depending on where translation begins, and three proteins are synthesized (see FIG. 12). In this way, in principle, three reading frames and the synthesis of three proteins based on the reading frames can be considered. However, in most cases, a functional protein is inserted due to a stop codon in the middle. It is said that only one way can be synthesized.
[0101]
The translation process is explained above. However, for some reason in the translation process, one base of RNA may be skipped, or one base may be read twice, resulting in a frame shift phenomenon.
[0102]
Therefore, it was considered that a phenomenon of +1 frame shift occurred due to the skipping of one base of the RNA portion between the two proteins (see FIGS. 13 and 14). The frame shift was predicted to occur around 250-290 bases where the two proteins overlap.
[0103]
On the other hand, there is a possibility that there is an error in the data stored in the database. Therefore, the entire base sequence of P. furiosus DNA was confirmed. Specifically, genomic DNA was collected from P. furiosus bacteria, and the base sequence of the target portion was read from the collected genomic DNA (see FIG. 15).
[0104]
The operation of collecting genomic DNA from P. furiosus bacteria was performed as follows. First, 5 ml of bacteria (P. furiosus) was collected and gently mixed with a DNA extraction buffer containing Proteinase K. Next, after incubation at 37 ° C. for 1 hour, NaCl and CTAB / NaCl solutions were mixed, and further incubated (65 ° C., 10 minutes). Next, it was treated with phenol / chloroform and a precipitate (genomic DNA) was obtained with ethanol. The “CTAB” is an abbreviation for cetyltrimethylammonium bromide.
[0105]
Next, partial DNA was amplified using the genomic DNA. Specifically, a DNA fragment containing a portion of a gene that seems to have a frame shift was amplified by a PCR (Polymerase chain reaction) method.
[0106]
Here, the principle of the PCR method will be briefly described. The principle of the PCR method is to repeatedly perform a DNA synthesis reaction consisting of the following three steps.
(Step 1) The template DNA is converted to a single strand by denaturation by heating.
(Step 2) The temperature is lowered in a state where two kinds of oligonucleotide primers complementary to both ends of the DNA strand to be amplified (hereinafter, appropriately referred to as “primers”) are added excessively to the reaction system. By this operation, the primer forms a double strand with a portion complementary to the template DNA strand.
(Step 3) In the state of Step 2, the synthetic substrate dNTP (deoxyribonucleotide triphosphate) and DNA polymerase are allowed to act. By this operation, a complementary strand of DNA is synthesized from the portion where the primer forms a double strand.
[0107]
The DNA synthesized in the first cycle serves as a template for the next reaction. Therefore, DNA is synthesized in a chain reaction after the next cycle. Then, after 30 cycles of reaction, enormous DNA molecules are obtained.
[0108]
Next, a specific operation method will be described. First, the reaction liquid shown below was mixed. Next, it was set in a thermal cycler to amplify the DNA. As the template DNA, the extracted genomic DNA of P. furiosus was used. Primers were prepared based on the base sequence of P. furiosus on the database so that the obtained DNA would be about 240 bases including the frameshift portion. In this reaction (PCR), Takara Taq ^TM (Takara Shuzo) was used.
[0109]
The composition of the reaction solution in the PCR reaction is genomic DNA (1 μl), primer (1 μl), LATaq (1 μl), buffer (5 μl), dNTP (5 μl), and H ₂ O (36 μl). The primers used for the PCR reaction are shown below.
(Primer)
5 'AAGGGAGGAGAAGGCTCACG 3'
3 'TTCTCGTTACTGGCAAGTGC 5'
The temperature conditions in the PCR reaction are shown in Steps 1 to 5 below. The temperature condition of the PCR reaction started from Step 1, and after Steps 2 to 4 were performed for 30 cycles, proceeded to Step 5.
Step 1 95 ° C (2 minutes)
Step 2 94 ° C (30 seconds)
Step 3 55 ° C (1 minute)
Step 4 72 ° C (2 minutes)
Step 5 72 ° C (10 minutes)
The PCR product obtained by the above operation was subjected to electrophoresis on a 0.7% agarose gel. The result is shown in FIG. As shown in FIG. 16, amplification of DNA having a target portion of about 230 to 240 bases was confirmed by electrophoresis.
[0110]
Next, the base sequence of the amplified DNA was confirmed. A DNA sequencer was used for the confirmation. The DNA sequencer analyzes four-color fluorescence-labeled DNA fragments grown by PCR at four-color wavelengths. A fluorescently labeled target DNA fragment is prepared by adding a primer and a fluorescent reagent and performing PCR. In this example, the DNA base sequence was confirmed using a BigDye-Terminator Cycle Sequencing kit (Applied Biosystems).
[0111]
The composition of the reaction solution in PCR for confirming the base sequence of DNA is Premix (4 μl), buffer (4 μl), primer (3.2 pmol), and template DNA (300 ng). The PCR reaction conditions (temperature conditions) are the following steps 1 to 3 (25 cycles).
Step 1 96 ° C (20 seconds)
Step 2 50 ° C (10 seconds)
Step 3 60 ℃ (4 minutes)
Next, a PCR product was obtained by precipitation with ethanol. Then, Template Suppression Reagent was added and heated to 95 ° C. for 2 minutes, and then cooled on ice. The DNA sequencer (ABI PRISM (registered trademark) 310 Gentetic Analyzer (Applied Biosystems)) was set and analyzed.
[0112]
By comparing the base sequence of the DNA obtained by the above confirmation with the base sequence in the database one by one, it is confirmed that one of the base sequences in the database (the 271st base “c”) is missing. (See FIG. 17). This indicates that the P. furiosus gene data stored in the database was incorrect. In other words, the frame shift is not performed in the middle of translation, but the database data has an extra base, so one protein is originally interpreted as having two proteins on the database. (See FIG. 18). FIG. 18 shows a corrected base sequence and a part of the corresponding amino acid sequence. Note that “TELKGGFEEV” shown in bold indicates the protein PF1192 in the P. furiosus database. Further, “KELTGIEAH” shown in bold and italic letters indicates the protein PF1191 in the P. furiosus database.
[0113]
As described above, it was found that there is a difference between the data in the database and the actual DNA base sequence. Therefore, the primer design was changed, and all 1038 bases encoding the shell protein were cloned to confirm the base sequence.
[0114]
The base sequence was confirmed by first amplifying the target portion from the genomic DNA of P. furiosus by PCR and confirming the amplification of the sequence of the target portion by electrophoresis. Then, after the amplified product was incorporated into a vector (pTA), the DNA base sequence was confirmed.
[0115]
The PCR reaction solution is genomic DNA (1 μl), buffer (5 μl), primer (1 μl), dNTP (5 μl), LATaq (1 μl), and Mg buffer (5 μl). The reaction conditions (temperature conditions) used for the PCR are shown in Steps 1 to 5 below. The reaction conditions for the PCR reaction started from Step 1, and after Steps 2 to 4 were performed 30 cycles, proceeded to Step 5.
Step 1 95 ° C (2 minutes)
Step 2 94 ° C (30 seconds)
Step 3 56 ° C (1 minute)
Step 4 72 ° C (2 minutes)
Step 5 72 ° C (10 minutes)
The primers used for the PCR reaction are shown below.
(Primer)
N: 5 'GATCCATATGCTCTCAATAAATCC 3'
C: 5 'ATGCACATATGGCCGTGAAC 3'
Further, as described above, the nucleotide sequence was confirmed after incorporation into the pTA vector. In the PCR reaction at the time of confirmation (sequence) of the base sequence, since the DNA length exceeds 1000 bases, the primer was divided into two, and the base sequence of the complementary strand was also confirmed. In this primer design, primers N and C were used before and after insertion of the pTA vector. Then, DNAs located about half of the target portion were used as primers M1 and M2. That is, the base sequence was confirmed using a total of four primers, primers N and M1 and primers C and M2. These primers are shown below.
(Primer)
N: 5 'GATCCATATGCTCTCAATAA 3'
C: 5 'ATGCACATATGGCCGTGAA 3'
M1: 5 'GAAGAAGAAATTCTATGTGG 3'
M2: 5 'CCACATAGAATTTCTTCTTC 3'
The reaction conditions (temperature conditions) of the PCR reaction at the time of confirming the base sequence are shown in the following Step 1 to Step 3. In addition, the reaction conditions of this PCR reaction performed 25 cycles of steps 1-3.
Step 1 96 ° C (20 seconds)
Step 2 50 ° C (10 seconds)
Step 3 60 ℃ (4 minutes)
The reaction solution of the PCR reaction at the time of confirming the base sequence is a circular DNA (4 μl) incorporating DNA, a buffer (4 μl), a primer (4 μl), H ₂ O (6 μl).
[0116]
As a result, the other sequences were the same as those in the database. From the above, the entire nucleotide sequence and the entire amino acid sequence of the shell protein of the virus-like particle were determined. The complete nucleotide sequence and the entire amino acid sequence are shown in FIG. The entire amino acid sequence of the virus-like particle shell protein is also shown in SEQ ID NO: 3 in the sequence listing. Further, the entire base sequence encoding the entire amino acid sequence of the shell protein is also shown in SEQ ID NO: 1 in the sequence listing. The sequence shown in SEQ ID NO: 2 is a sequence in which the base sequence shown in SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 3 are written together.
[0117]
Initially, virus-like particles were thought to be derived from foreign species, so if there is a match in the database of amino acid sequences and nucleotide sequences, it matches the database of other viruses, not the database of P. furiosus I was expecting that. Or he thought that a new virus that had not yet been discovered would not have been registered in the database and would not have a search match.
[0118]
However, according to the above results, it was found that the origin of the virus-like particles is in the species of P. furiosus itself. This was a new fact that the amino acid sequence of the shell protein constituting the virus-like particle was encoded on the gene of P. furiosus. That is, this virus-like particle may be originally expressed in P. furiosus.
[0119]
Example 5 Amino Acid Sequence Homology
Next, from the homology of the determined amino acid sequence, how this virus-like particle exists in the host P. furiosus and whether the virus-like particle exists in other species. Verified.
[0120]
Homology is the similarity found in a progeny gene branched from a common ancestor gene by speciation or gene duplication or its product. Searching for similar sequences by comparing a specific base sequence or amino acid sequence (query sequence) with all sequences registered in the database is called homology search. In the homology search, when a significant similarity (about 20% or more) with the amino acid sequence of a known protein is detected, it is considered that the structure / function is the same.
[0121]
First, using ClustalW sequence analysis on the web (http://clustalw.genome.ad.jp), sequence analysis is performed by performing multiple alignment with bacteriophage HK97 shell protein having structural similarity. (See Nucleic Acids Res 22 4673-4680. (1994)). And it was investigated using the FASTA database (http://www.fasta.genome.ad.jp) whether there was a protein similar to the amino acid sequence of 40 kDa protein (shell protein which comprises virus-like particle | grains). (See Proc Natl Acad Sci USA 85 (8) 2444-8 (1988)).
[0122]
According to the analysis of the sequence by the multiple alignment, the similarity to bacteriophage HK97 was 19%. In addition, the proteins that are considered to have high similarity found by the investigation using the FASTA database are the four proteins shown in the following Table 1 (No. 2 to 5 proteins in the table), that is, the similarity is 20% or more. It is a protein.
[0123]
[Table 1]

[0124]
In the proteins of the genus Sulfolobus shown in No. 2 and No. 3 in Table 1, the length (number of amino acid residues) of the amino acid sequence hardly changed (about 340 amino acid residues), and the similarity was about 30%. The proteins shown in No. 4 and No. 5 in Table 1 are proteins of the same Pyrococcus genus, and although the length of the protein is as short as 101 residues, it showed a high similarity of 50% or more. Since the proteins other than the above four types have different protein lengths and a similarity of less than 20%, it was judged that there is no homology.
[0125]
FIG. 20 shows a comparison result between the amino acid sequence of Sulfolobus tokodaii ST 0992 shown in No. 2 of Table 1 and the amino acid sequence of P. furiosus. As shown in Table 1, the similarity is 30%, but according to FIG. 20, it can be seen that the similarity in the first half is higher than the similarity in the second half.
[0126]
In FIG. 20 and FIG. 21 described later, “*”, “:”, “.”, And “” (blank) are used as symbols indicating homology. “*” Indicates that the amino acid sequence is completely conserved between species. “:” Indicates that a mutation between amino acid residues having very close properties occurs between species. “.” Indicates that variation between amino acid residues having similar properties occurs between species. “” (Blank) indicates that a mutation that cannot be represented by “:” and “.” Occurs between species.
[0127]
In the case of bacteriophage HK97, the similarity was expected to be considerably high because there is a portion where the steric structure is similar. However, actually, as shown in Table 1, the similarity of amino acid sequences was not so high as 19%. From this, it is presumed that the shell protein has a sufficient possibility of becoming a virus-like particle even if it is not so similar.
[0128]
All of the four proteins found in the homologous sequence (proteins of numbers 2 to 5 in Table 1) are proteins whose functions are unknown. The genus Sulfolobus, which is a species of the protein shown in numbers 1 to 3 in Table 1, is a thermophilic thermophile classified as Crenarchaeota (one of archaeal subdomains) and grows even at 100 ° C or higher. But not close to P.furiosus. However, the similarity of the protein of interest (the proteins shown in Tables 1 and 2) has about 30% similarity. This number is higher than the similarity of HK97 to the shell protein, as shown in Table 1. Furthermore, the number of amino acid residues of the proteins shown in Nos. 2 to 3 in Table 1 is very close to the number of amino acids of the shell protein (345 residues) constituting the virus-like particle analyzed by the inventors. From this, it is presumed that the proteins shown in Nos. 2 to 3 in Table 1 are proteins having the same function and structure as the P. furiosus shell protein, and the virus-like particles analyzed by this inventor are of the genus Sulfolobus. It can be said that there is also a possibility.
[0129]
Pyrococcus abyssi and Pyrococcus horikoshii, which are protein species shown in numbers 4 to 5 in Table 1, are the same Pyrococcus genus as P. furiosus, have similar fungal growth conditions, and the similarity of each protein is also very high. Both of the proteins found by this similarity search are 101 residues, which are different from the 345 residues of the protein constituting the virus-like particle discovered by the inventors. However, the result was that the similarity was more than 50% only in the first half from the N-terminal. FIG. 21 shows the homology between the amino acid sequence of the P. furiosus shell protein and the amino acid sequence of Pyrococcus horikoshii PH1734, showing that there is a similarity of 53%. The absence of a highly similar portion of about 250 residues in the latter half of the genus Pyrococcus can be said to have been deceived and lost for some reason during the evolution process.
[0130]
Moreover, from the result of the analyzed three-dimensional structure of the virus-like particle and the result of this homology search, the first half and the second half of the shell protein constituting the virus-like particle function as if they are different proteins.・ The structure is considered to be separated. This is considered for the following reason.
[0131]
(Reason 1: structural reason) In the monomer structure of virus-like particles, the portion constituting the shell of virus-like particles is only about 250 residues in the latter half, and about 100 residues from the N-terminal in the first half. Is considered to be swayed in the protein structure inside the particle. Therefore, the first 100 residues cannot be confirmed by the electron density map. FIG. 22 shows the monomer structure of the virus-like particle, indicating that the first 100 residues cannot be confirmed in the latter 250-residue ribbon diagram.
[0132]
(Reason 2: Reasons based on homology search results) In the homology search, when comparing carefully with the amino acid sequence of the protein of the genus Sulfolobus, the similarity of the sequence of the first 100 residues is 37%, the latter 250 residues The sequence similarity is 20%, and the homology is different between the first half and the second half. Furthermore, as described in Table 1, P. abysii and P. horikoshii do not have a similar sequence of the latter 250 residues, although the sequence similarity of the first 100 residues exceeds 50%. In other words, the first half and the second half are also separated from the similarity.
[0133]
Therefore, there is a possibility that the latter 250-residue part constitutes the shell of the virus-like particle, and the first 100-residue part is a functional part that recognizes nucleic acids and the like inside the virus. Is strong. From this, it is considered that even in the same genus Pyrococcus, only a protein derived from P. furiosus becomes a virus-like particle, and a protein derived from P. abysii and P. horikoshii, which has only a protein in the first half, does not become a particle.
Example 6 Expression System Using E. coli
As an application of this thermostable virus-like particle, an expression system using E. coli was constructed. First, proteins constituting virus-like particles were expressed using pET9 and pET11 as expression vectors and BL211 (DE3) pLysS and BL21 (DE3) Codon + as hosts. Next, the expression of the target protein was confirmed by SDS-PAGE. In addition, the protein was purified by centrifugation. The purification was performed by first crushing the cells with ultrasonic waves, followed by heat treatment (80 ° C., 1 hour). Next, after gel filtration with Sephadex200, the protein was purified by sucrose discontinuous density gradient centrifugation (40-10%, 20000 rpm, 10 hours). Then, after protein purification, the formation of a spherical shell structure was confirmed by an electron microscope.
[0134]
It has been confirmed that a spherical shell structure is formed even under conditions of an expression system using E. coli. The conditions are Tris-HCl (pH 7.5) (20 mM), glycerol (10%), EDTA (5 mM), NaCl (100 mM).
[0135]
Thus, it was confirmed that the shell protein was expressed using E. coli and that a spherical shell structure was formed. This shows the possibility that virus-like particles (spherical particles) can be produced in large quantities using the gene and protein of the present invention.
[0136]
The following can be said by Examples 1-6.
(1) The virus-like particle has a diameter of 30 nm.
(2) Virus-like particles exist in a hyperthermophilic bacterium P. furiosus while symbiotic.
(3) The shell protein of virus-like particles has two functions and structures.
(4) A spherical structure can be formed by an expression system using Escherichia coli.
[0137]
From homology search, similar virus-like particles may exist in two species of the genus Sulfolobus, and only those derived from P. furiosus are expected to be virus-like particles in the same genus Pyrococcus. Is done. Therefore, it turned out that virus-like particles are also interesting objects in evolution.
[0138]
【The invention's effect】
As described above, the gene of the present invention encodes a protein constituting a spherical particle. Therefore, it is possible to easily synthesize a protein constituting the spherical particle by this gene, and further to produce a virus-like particle (spherical particle) using the protein.
[0139]
Moreover, the protein of this invention is a protein which forms a spherical particle by gathering as mentioned above. In addition, the gene encoding the protein has been identified. Therefore, there is an effect that the production and use of spherical particles (virus-like particles) becomes easy.
[0140]
The spherical particles of the present invention are characterized by having the above protein. Therefore, there is an effect that spherical particles can be easily produced and used by a method of assembling the proteins.
[0141]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic view and a cross-sectional view showing the structure of virus-like particles (spherical particles) obtained by structural analysis.
FIG. 2 is a graph showing the relationship between the eluted fraction number and the absorbance in hydrophobic chromatography according to an example of the present invention.
FIG. 3 is a schematic diagram showing the results when SDS-PAGE was performed on the eluted fraction obtained by hydrophobic chromatography in the examples of the present invention.
FIG. 4 is a schematic diagram showing the results when SDS-PAGE is performed on a sample before crystallization, a sample separated by column chromatography, and a sample of a crystal solution.
FIG. 5 is an electron micrograph showing an observation result of a sample before crystallization.
FIG. 6 is an electron micrograph showing the observation results of a sample separated by hydrophobic column chromatography.
FIG. 7 is an electron micrograph showing an observation result of a sample of a crystal solution.
FIG. 8 is an explanatory diagram illustrating the form of a bacterial virus.
FIG. 9 is a schematic diagram showing the results of HPLC of the N-terminal sequencing method when P (proline) at the fifth residue is found as a result of analysis of the partial amino acid sequence.
FIG. 10 is a schematic diagram showing a result of analyzing a target protein after performing SDS-PAGE using a mass spectrometer.
FIG. 11 is a schematic diagram showing the amino acid sequences of proteins (PF1191 and PF1192) found in the P. furiosus database.
FIG. 12 is a schematic diagram showing three possible reading frames in protein synthesis.
FIG. 13 is a schematic diagram showing a base sequence of a target portion and a corresponding amino acid sequence based on database data and analysis results in an example of the present invention.
FIG. 14 is a schematic diagram showing a continuation of FIG. 13;
FIG. 15 is a schematic diagram showing that target DNA is amplified by PCR from genomic DNA of P. furiosus and the target amino acid sequence is read.
[Fig. 16] A primer prepared based on the base sequence of P. furiosus in the database so that the obtained DNA has about 240 bases including a frameshift portion, and genomic DNA of P. furiosus. It is a schematic diagram which shows the result when PCR is performed and the PCR product is subjected to electrophoresis.
FIG. 17 is a schematic diagram showing the results when comparing the base sequence of DNA obtained by analysis and the base sequence in the database one by one.
FIG. 18 is an explanatory diagram for explaining that two proteins (PF1191 and PF1192) are connected together with the corrected base sequence and a part of the corresponding amino acid sequence.
FIG. 19 is a diagram showing a corrected total base sequence of 1038 bases and a corresponding total amino acid sequence of 345 residues.
FIG. 20 is a schematic diagram showing homology between the amino acid sequence of Sulfolobus tokodaii ST 0992 and the amino acid sequence of P. furiosus.
FIG. 21 is a schematic view showing the homology between the amino acid sequence of P. furiosus shell protein and the amino acid sequence of Pyrococcus horikoshii PH1734.
FIG. 22 is a three-dimensional structural diagram showing the monomer structure of virus-like particles (the structure of the shell protein of virus-like particles).

Claims (7)

A gene encoding the following protein (a) or (b) :
(A) a protein comprising the amino acid sequence of SEQ ID NO: 3.
(B) of the amino acid sequence shown in SEQ ID NO: 3, one or several amino acids substitution, deletion, insertion, and / or added in the amino acid sequence, a protein which forms spherical particles by aggregation . Gene, comprising the nucleotide sequence of SEQ ID NO: 1. Of the nucleotide sequence set forth in SEQ ID NO: 1, consists of one or several bases are substituted, deleted, inserted, and / or added in the nucleotide sequence,
A gene that encodes a protein that forms a spherical particle when assembled. The protein, the gene of claim 3, characterized in that the amino acid sequence set forth in SEQ ID NO: 3. The protein is the amino acid sequence set forth in SEQ ID NO: 3, one or several amino acids are substituted, deleted, inserted, and / or claim 3, characterized in that it consists added in the amino acid sequence Genes. Proteins comprising an amino acid sequence set forth in SEQ ID NO: 3. Of the amino acid sequence shown in SEQ ID NO: 3, made of one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence,
A protein characterized by forming spherical particles by assembling.

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