JP6379537B2 - Recombinant hydrogen-oxidizing bacteria and protein production method using the same - Google Patents
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本発明は、目的タンパク質遺伝子を導入することで前記タンパク質を発現可能な水素酸化細菌、およびそれを用いた前記タンパク質の製造方法に関する。 The present invention relates to a hydrogen-oxidizing bacterium capable of expressing the protein by introducing a target protein gene, and a method for producing the protein using the same.
近年、微生物や細胞を用いたタンパク質の生産研究が盛んである。特に、分子量が大きく、かつ複雑な構造を有したタンパク質を含むバイオ医薬品は、低分子医薬品と比べ標的分子に対する高い特異性と親和性を有することから、従来の医薬品では対応できなかった疾患に対応しうる医薬として将来需要が増大することが予測される。 In recent years, protein production research using microorganisms and cells has been actively conducted. In particular, biopharmaceuticals that include proteins with large molecular weights and complex structures have higher specificity and affinity for target molecules than low-molecular-weight drugs, so they can handle diseases that cannot be handled by conventional drugs. It is predicted that future demand will increase as a possible medicine.
バイオ医薬品に用いられるタンパク質を工業的に生産する場合、主に遺伝子組み換え技術や細胞培養技術を用いて生産され、その多くは大腸菌を宿主とした生産系を利用している。しかしながら大腸菌を宿主とした従来の生産系では、培養の際、培地中に酵母エキス、ペプトン、アミノ酸、ビタミンなどの高価な原料を用いる必要がある上、目的タンパク質の生産量が低いため、製造コストが高くなるという問題点がある。また前記生産系の多くは目的タンパク質を大腸菌内に発現させており、大腸菌体内から目的タンパク質を抽出する操作が必要である。さらに生産した目的タンパク質が大腸菌体内由来のタンパク質分解酵素で分解され純度が低下する問題や、形成した封入体のリフォールディングによる精製コストの上昇の問題もある。 When proteins used for biopharmaceuticals are industrially produced, they are mainly produced using genetic recombination techniques and cell culture techniques, and most of them use production systems using E. coli as a host. However, in the conventional production system using E. coli as a host, it is necessary to use expensive raw materials such as yeast extract, peptone, amino acids and vitamins in the culture medium, and the production cost of the target protein is low. There is a problem that becomes high. Many of the production systems express the target protein in E. coli, and an operation for extracting the target protein from the E. coli body is required. In addition, there is a problem that the produced target protein is degraded by a proteolytic enzyme derived from the body of Escherichia coli and the purity is lowered, and the purification cost is increased due to refolding of the formed inclusion bodies.
酵母や枯草菌を宿主として用いた生産系は、目的タンパク質を培養液中に分泌発現させることが可能である。しかしながら大腸菌を宿主とした系と同様、酵母エキスなどの高価な原料を用いる必要がある。また宿主由来のタンパク質分解酵素による目的タンパク質の分解や、培地中に含まれる窒素源由来の副生産物による精製コストの上昇の問題もあった。 A production system using yeast or Bacillus subtilis as a host can secrete and express the target protein in the culture medium. However, it is necessary to use expensive raw materials such as yeast extract as in the system using E. coli as a host. There are also problems of degradation of the target protein by a host-derived proteolytic enzyme and an increase in purification cost due to a by-product derived from a nitrogen source contained in the medium.
大腸菌、酵母、枯草菌以外に物質生産において有効と考えられる宿主のひとつとして、水素酸化細菌Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａが知られている。Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａは窒素、リン、酸素などが供給制限となるような条件下で大量のＰＨＢ（ポリヒドロキシ酪酸）を蓄積することが報告されており、従属栄養条件下の高密度培養ではＰＨＢを含む菌体収量は２８１ｇ／Ｌにまで達することが確認されている（非特許文献１）。また、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａは水素酸化によるエネルギーで炭素固定を行なう独立栄養条件でも増殖できるほか、無機塩と糖のみを原料とした培養でも高密度培養が可能であることが知られている。これまで、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａを宿主とした物質生産は、前述したＰＨＢのほかにも、２−メチルクエン酸（非特許文献２）、２−ＨＩＢＡ（ヒドロキシイソブチル酸）（非特許文献３）、Ｒ−３−ヒドロキシ酪酸（非特許文献４）、シアノフィシン（非特許文献５）などの例が報告されている。また無機塩とグルコースのみの原料からタンパク質である有機リン分解酵素を大量生産した例も報告されている（非特許文献６および７）。
In addition to Escherichia coli, yeast, and Bacillus subtilis, a hydrogen-oxidizing bacterium Ralstonia eutropha is known as one of the hosts considered to be effective in substance production. Ralstonia eutropha has been reported to accumulate a large amount of PHB (polyhydroxybutyric acid) under conditions where supply of nitrogen, phosphorus, oxygen, etc. is restricted, and bacteria containing PHB in high-density culture under heterotrophic conditions It has been confirmed that the body yield reaches 281 g / L (Non-patent Document 1). In addition, it is known that Ralstonia eutropha can grow under autotrophic conditions in which carbon fixation is performed by energy generated by hydrogen oxidation, and high-density culture is possible even in culture using only inorganic salts and sugars. Up to now, substance production using Ralstonia eutropha as a host is not limited to PHB described above, but also 2-methylcitric acid (Non-patent Document 2), 2-HIBA (hydroxyisobutyric acid) (Non-patent Document 3), R- Examples such as 3-hydroxybutyric acid (Non-Patent Document 4) and cyanophycin (Non-Patent Document 5) have been reported. In addition, examples of mass production of protein-derived organophosphorus degrading enzymes from raw materials consisting only of inorganic salts and glucose have been reported (Non-patent
水素酸化細菌Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａに代表されるＲａｌｓｔｏｎｉａ属の細菌はもともと温泉や土壌など様々な環境に生息している微生物であり、独立栄養条件で増殖できるなど、その環境適応性から物質の取り込みに関わる機能が発達していることが示唆されてきた。実際、芳香族化合物の取り込みや分解を行うＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＪＭＰ１３４、重金属アンチポーターによる重金属排出能を持つＲａｌｓｔｏｎｉａ ｍｅｔａｌｌｉｄｕｒａｎｓ ＣＨ３４、多数の細胞外毒素を放出する機構を持つＲａｌｓｔｏｎｉａ ｓｏｌａｎａｃｅａｒｕｍ、細胞外ＰＨＢデポリメラーゼを持つＲａｌｓｔｏｎｉａ ｐｉｃｋｅｔｔｉｉなどの様々な種が存在し、細胞膜上の物質輸送に関するタンパク質が多様性に富んでいることが明らかとなっている。また、ゲノム情報解析の結果では、ゲノム中のタンパク質遺伝子のうち１２％が物質の輸送に関わるものであり、大腸菌の９％と比べ多く、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ属の細菌における膜輸送タンパク質の多様性を裏付ける結果となっている。しかしながらこれまで、Ｒａｌｓｔｏｎｉａ属などの水素酸化細菌の輸送タンパク質を利用した水素酸化細菌を宿主とするタンパク質の分泌発現系の報告はなかった。 Bacteria belonging to the genus Ralstonia, represented by the hydrogen-oxidizing bacterium Ralstonia eutropha, are microorganisms that originally inhabit various environments such as hot springs and soils, and can grow under autotrophic conditions. Has been suggested to have developed. In fact, Ralstonia eutropha JMP134, which takes up and degrades aromatic compounds, Ralstonia metallidurans CH34 with heavy metal excretion by heavy metal antiporters, Ralstonia solanacerum with a mechanism to release many extracellular toxins, Ralstonia with extracellular PHB depolymerase There are various species such as pickettii, and it is clear that proteins related to mass transport on the cell membrane are rich in diversity. In addition, as a result of genome information analysis, 12% of protein genes in the genome are involved in substance transport, which is more than 9% in E. coli, confirming the diversity of membrane transport proteins in bacteria belonging to the genus Ralstonia eutropha It is the result. However, there has been no report on a protein secretory expression system using a hydrogen-oxidizing bacterium as a host using a transport protein of a hydrogen-oxidizing bacterium such as Ralstonia.
そこで本発明の目的は、目的タンパク質遺伝子を導入した水素酸化細菌において、前記目的タンパク質を菌体外へ分泌発現可能な水素酸化細菌、および前記水素酸化細菌を用いた目的タンパク質の製造方法を提供することにある。 Accordingly, an object of the present invention is to provide a hydrogen-oxidizing bacterium capable of secreting and expressing the target protein outside the cell in a hydrogen-oxidizing bacterium into which the target protein gene has been introduced, and a method for producing the target protein using the hydrogen-oxidizing bacterium. There is.
本発明者は、上記課題を解決するため鋭意検討を重ねた結果、目的タンパク質遺伝子を導入した水素酸化細菌において、輸送タンパク質遺伝子をさらに導入し、前記水素酸化細菌により前記目的タンパク質と前記輸送タンパク質とを共発現させることで、目的タンパク質を培地中（水素酸化細菌外）に効率よく分泌発現できることを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above problems, the present inventor further introduced a transport protein gene in the hydrogen-oxidizing bacterium into which the target protein gene has been introduced, and the target protein and the transport protein are separated by the hydrogen-oxidizing bacterium. By co-expressing the protein, the inventors have found that the target protein can be efficiently secreted and expressed in the medium (outside of hydrogen-oxidizing bacteria), and the present invention has been completed.
すなわち本発明は以下の態様を包含する。 That is, this invention includes the following aspects.
（１）目的タンパク質遺伝子および輸送タンパク質遺伝子を水素酸化細菌に導入して得られる、前記目的タンパク質を発現可能な水素酸化細菌。 (1) A hydrogen-oxidizing bacterium capable of expressing the target protein obtained by introducing a target protein gene and a transport protein gene into the hydrogen-oxidizing bacterium.
（２）輸送タンパク質が配列番号７または１０に記載の配列からなるポリペプチドである、（１）に記載の水素酸化細菌。 (2) The hydrogen-oxidizing bacterium according to (1), wherein the transport protein is a polypeptide having the sequence described in SEQ ID NO: 7 or 10.
（３）輸送タンパク質遺伝子が配列番号１１または１２に記載の配列からなるポリヌクレオチドである、（１）に記載の水素酸化細菌。 (3) The hydrogen-oxidizing bacterium according to (1), wherein the transport protein gene is a polynucleotide comprising the sequence described in SEQ ID NO: 11 or 12.
（４）水素酸化細菌がＲａｌｓｔｏｎｉａ属細菌である、（１）から（３）のいずれかに記載の水素酸化細菌。 (4) The hydrogen-oxidizing bacterium according to any one of (1) to (3), wherein the hydrogen-oxidizing bacterium is a genus Ralstonia.
（５）目的タンパク質がＦｃ結合性タンパク質である、（１）から（４）のいずれかに記載の水素酸化細菌。 (5) The hydrogen-oxidizing bacterium according to any one of (1) to (4), wherein the target protein is an Fc-binding protein.
（６）（１）から（５）のいずれかに記載の水素酸化細菌を培養し、目的タンパク質および輸送タンパク質を共発現させることで、前記水素酸化細菌外へ目的タンパク質を分泌発現させる方法。 (6) A method for secreting and expressing a target protein outside the hydrogen-oxidizing bacterium by culturing the hydrogen-oxidizing bacterium according to any one of (1) to (5) and co-expressing the target protein and a transport protein.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明において、宿主として用いる水素酸化細菌は特に限定はないものの、独立栄養条件下で増殖でき、かつ物質の取り込みに関わる機能が発達しているＲａｌｓｔｏｎｉａ属細菌が好ましく、その中でもＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａは多様な環境適応性を持ち、かつ物質輸送に関わるタンパク質の機能や数が豊富である点で、特に好ましい水素酸化細菌といえる。 In the present invention, the hydrogen-oxidizing bacterium used as a host is not particularly limited, but Ralstonia genus bacteria that can grow under autotrophic conditions and have developed functions related to substance uptake are preferable. Among them, Ralstonia eutropha is various. It can be said to be a particularly preferred hydrogen-oxidizing bacterium because it has environmental adaptability and has abundant functions and numbers of proteins involved in mass transport.
本発明は、水素酸化細菌を宿主として目的タンパク質を発現させる際に、目的タンパク質遺伝子に加えて輸送タンパク質遺伝子も水素酸化細菌に導入することを特徴としている。輸送タンパク質は、水素酸化細菌が有する分泌性タンパク質または膜タンパク質の中から、目的タンパク質との共発現により当該目的タンパク質を培地中に放出する機能を持つタンパク質を適宜選択すればよいが、親水性の高いタンパク質や分子量４０ｋＤａ以下ののタンパク質を選択すると好ましい。なお輸送タンパク質の全長が４０ｋＤａ以上の場合は、親水性の高い領域のみを利用し４０ｋＤａ以下の輸送タンパク質として発現させればよい。輸送タンパク質の一例としては、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａの染色体１由来のＨ１６＿２６２０（配列番号７）やＨ１６＿Ａ０９８３のＮ末端側領域（配列番号１０）があげられる。
The present invention is characterized in that when a target protein is expressed using a hydrogen-oxidizing bacterium as a host, a transport protein gene is also introduced into the hydrogen-oxidizing bacterium in addition to the target protein gene. As the transport protein, a protein having a function of releasing the target protein into the medium by co-expression with the target protein may be appropriately selected from secretory proteins or membrane proteins possessed by hydrogen-oxidizing bacteria. It is preferable to select a high protein or a protein having a molecular weight of 40 kDa or less. In addition, when the full length of transport protein is 40 kDa or more, what is necessary is just to express as a transport protein of 40 kDa or less using only a highly hydrophilic area | region. Examples of transport proteins include H16_2620 (SEQ ID NO: 7) derived from
水素酸化細菌に導入する輸送タンパク質遺伝子は、前記輸送タンパク質のｃＤＮＡ等からＰＣＲ法などのＤＮＡ増幅法を用いて調製後適当な方法で連結して得てもよいし、前記輸送タンパク質のアミノ酸配列からヌクレオチド配列に変換後人工的に合成して得てもよい。アミノ酸配列からヌクレオチド配列に変換する際は、形質転換させる宿主におけるコドンの使用頻度を考慮して変換するのが好ましい。コドンの使用頻度の解析は公的データベース（例えば、かずさＤＮＡ研究所のホームページにあるＣｏｄｏｎ Ｕｓａｇｅ Ｄａｔａｂａｓｅなど）を利用することによっても可能である。輸送タンパク質遺伝子の一例として、配列番号７に記載のアミノ酸配列からなる輸送タンパク質（Ｈ１６＿２６２０）をコードするポリヌクレオチドである配列番号１１に記載の配列からなるポリヌクレオチドや、配列番号１０に記載のアミノ酸配列からなる輸送タンパク質（Ｈ１６＿Ａ０９８３のＮ末端側領域）をコードするポリヌクレオチドである配列番号１２に記載の配列からなるポリヌクレオチドがあげられる。 The transport protein gene to be introduced into the hydrogen-oxidizing bacterium may be obtained by preparing the transport protein cDNA or the like using a DNA amplification method such as a PCR method after ligation by an appropriate method, or from the amino acid sequence of the transport protein. It may be obtained by artificial synthesis after conversion to a nucleotide sequence. When converting from an amino acid sequence to a nucleotide sequence, it is preferable to convert in consideration of the codon usage in the host to be transformed. Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute). As an example of the transport protein gene, a polynucleotide consisting of the sequence described in SEQ ID NO: 11, which is a polynucleotide encoding the transport protein (H16_2620) consisting of the amino acid sequence described in SEQ ID NO: 7, or the amino acid sequence described in SEQ ID NO: 10 And a polynucleotide having the sequence set forth in SEQ ID NO: 12, which is a polynucleotide encoding a transport protein (N-terminal region of H16_A0983).
本発明において目的タンパク質遺伝子および輸送タンパク質遺伝子を導入する際、ペリプラズム中または外膜中に存在する宿主由来のプロテアーゼにより自動的に切断される適当な切断リンカーを介して連結し、目的タンパク質と輸送タンパク質との融合タンパク質として共発現させると好ましい。このようにすることで、発現した目的タンパク質と輸送タンパク質の融合タンパク質がペリプラズムに輸送された後プロテアーゼにより切断されることで、他の特別な処理を行なうことなく自動的に培地中に分泌されるからである。ここで用いる切断リンカーは、宿主由来のプロテアーゼで切断されるものであれば特に限定はなく、例えば大腸菌のｐｅｌＢやＰａｕｃｉｍｏｎａｓ ｌｅｍｏｉｇｎｅｉの細胞外ＰＨＢ分解酵素のシグナル配列であるｐｒｅＰｈａＺ１が例示できる。また本発明において目的タンパク質遺伝子および輸送タンパク質遺伝子を導入する際、（目的タンパク質遺伝子−（好ましくは切断リンカー）−輸送タンパク質遺伝子）の融合タンパク遺伝子の上流にファジンプロモーターを導入するとさらに好ましい。ファジンプロモーターはＰＨＢ（ポリヒドロキシ酪酸）顆粒の表面を覆うタンパク質であるファジン生産のプロモーターであり、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａの中で強力なプロモーターとして知られている。またファジンプロモーターは、培地中の窒素源やリン源の枯渇により活性化するため、大腸菌発現系などで用いられるＩＰＴＧ（イソプロピル−β−チオガラクトピラノシド）などの高価な誘導剤の添加なしにタンパク質の高発現が可能となる。 When introducing the target protein gene and transport protein gene in the present invention, the target protein and transport protein are linked via an appropriate cleavage linker that is automatically cleaved by a host-derived protease present in the periplasm or outer membrane. Co-expressed as a fusion protein with In this way, the fusion protein of the expressed target protein and transport protein is transported to the periplasm and then cleaved by the protease, so that it is automatically secreted into the medium without any other special treatment. Because. The cleavage linker used here is not particularly limited as long as it is cleaved by a host-derived protease, and examples thereof include prePhaZ1, which is a signal sequence of extracellular PHB-degrading enzyme of pelB of E. coli or Paucimonas lemignei. In addition, when introducing the target protein gene and the transport protein gene in the present invention, it is more preferable to introduce a phasin promoter upstream of the fusion protein gene of (target protein gene- (preferably cleavage linker) -transport protein gene). The phasin promoter is a promoter for producing phasin, which is a protein that covers the surface of PHB (polyhydroxybutyric acid) granules, and is known as a strong promoter in Ralstonia eutropha. In addition, since the fadin promoter is activated by depletion of nitrogen and phosphorus sources in the medium, it is possible to add an expensive inducer such as IPTG (isopropyl-β-thiogalactopyranoside) used in an E. coli expression system or the like. High protein expression is possible.
本発明の水素酸化細菌を作製する際、目的タンパク質遺伝子および輸送タンパク質遺伝子の水素酸化細菌への導入方法としては、主に広域宿主ベクターを用いた発現方法と自殺ベクターを用いたゲノム組換え法がある。水素酸化細菌としてＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａを用いた場合、広域宿主ベクターとしてはｐＢＢＲ１ＭＣＳ２、ｐＫＴ２３０、ｐＢＨＲ１が、自殺ベクターとしてはｐＪＱ２００ｍｐ１８、ｐＮＨＧ１、ｐＬＯ１が、それぞれ例示できる。なお広域宿主ベクターと自殺ベクターを併用して遺伝子導入を行なってもよい。 When producing the hydrogen-oxidizing bacterium of the present invention, the target protein gene and the transport protein gene are introduced into the hydrogen-oxidizing bacterium mainly by an expression method using a broad host vector and a genome recombination method using a suicide vector. is there. When Ralstonia eutropha is used as a hydrogen-oxidizing bacterium, pBBR1MCS2, pKT230, and pBHR1 can be exemplified as broad-area host vectors, and pJQ200mp18, pNHG1, and pLO1 can be exemplified as suicide vectors. In addition, gene transfer may be performed using a broad-area host vector and a suicide vector in combination.
本発明の水素酸化細菌は、大腸菌の発現系などと比べ、無機塩などの安価な原料からタンパク質の生産が可能である。例えば、大腸菌の培養で通常用いられる酵母エキスやペプトンなどの有機窒素原料を、アンモニア、アンモニウム塩、硝酸塩、亜硝酸塩などの無機窒素原料で置き換えることが可能である。また炭素源としては、グルコン酸やフルクトースの代わりに二酸化炭素や炭酸塩などの無機窒炭素原料で置き換えることが可能である。このような夾雑タンパク質のない無機塩原料の使用および目的タンパク質の培地中への分泌発現により、培養工程のみで培養上清中に高純度なタンパク質を得ることが出来、一般のタンパク質精製における煩雑な工程を簡素化できる。 The hydrogen-oxidizing bacterium of the present invention can produce proteins from inexpensive raw materials such as inorganic salts as compared to the expression system of Escherichia coli. For example, organic nitrogen materials such as yeast extract and peptone that are commonly used in E. coli culture can be replaced with inorganic nitrogen materials such as ammonia, ammonium salts, nitrates, and nitrites. Moreover, as a carbon source, it can replace with inorganic nitrogen carbon raw materials, such as a carbon dioxide and carbonate, instead of gluconic acid or fructose. By using such inorganic salt raw material without contaminating protein and secreting expression of the target protein into the medium, it is possible to obtain a high-purity protein in the culture supernatant only by the culturing process, which is complicated in general protein purification. The process can be simplified.
本発明の水素酸化細菌を用いて分泌発現可能な目的タンパク質に特に限定はなく、一例として、インシュリン、インターフェロン、インターロイキン、抗体、エリスロポエチン、成長ホルモンなどのヒト由来タンパク質、およびそれらの受容体タンパク質があげられる。なお本発明の水素酸化細菌を用いて分泌発現させる目的タンパク質は、完全体であってもよいし、目的タンパク質の機能に重要な部分のみから構成されるポリペプチドであってもよいし、さらに目的タンパク質を構成するアミノ酸の一つ以上が欠失および／または挿入および／または置換されていてもよい。以降、本発明の水素酸化細菌を用いて分泌発現可能な目的タンパク質のうち、Ｆｃ結合性タンパク質について詳細に説明する。 The target protein that can be secreted and expressed using the hydrogen-oxidizing bacterium of the present invention is not particularly limited, and examples include human-derived proteins such as insulin, interferon, interleukin, antibody, erythropoietin, growth hormone, and their receptor proteins. can give. The target protein to be secreted and expressed using the hydrogen-oxidizing bacterium of the present invention may be a complete protein, a polypeptide composed only of a part important for the function of the target protein, or a further object. One or more amino acids constituting the protein may be deleted and / or inserted and / or substituted. Hereinafter, among the target proteins that can be secreted and expressed using the hydrogen-oxidizing bacteria of the present invention, Fc-binding proteins will be described in detail.
本明細書においてＦｃ結合性タンパク質は、ヒトＦｃγＲＩの細胞外領域（具体的には天然型ヒトＦｃγＲＩの場合、配列番号１３に記載のアミノ酸配列のうち１６番目のグルタミンから２９２番目のヒスチジンまでの領域）、またはヒトＦｃγＲＩＩＩａの細胞外領域（具体的には天然型ヒトＦｃγＲＩＩＩａの場合、配列番号１４に記載のアミノ酸配列のうち１７番目のグリシンから２０８番目のグルタミンまでの領域）を構成するタンパク質のことをいう。ただし必ずしもヒトＦｃγＲＩ細胞外領域またはヒトＦｃγＲＩＩＩａ細胞外領域の全領域でなくてもよく、ヒトＦｃγＲＩ細胞外領域またはヒトＦｃγＲＩＩＩａ細胞外領域を構成するタンパク質（ポリペプチド）のうち、少なくとも抗体（免疫グロブリン）のＦｃ領域に結合する本来の機能を発現し得る領域のポリペプチドを含んでいればよい。当該Ｆｃ結合性タンパク質の一例として、
（Ｉ）配列番号１３に記載のアミノ酸配列のうち少なくとも１６番目のグルタミンから２８９番目のバリンまでのアミノ酸残基を含むタンパク質や、
（ＩＩ）配列番号１３に記載のアミノ酸配列のうち少なくとも１６番目のグルタミンから２８９番目のバリンまでのアミノ酸残基を含み、かつ前記アミノ酸残基のうちの一つ以上が他のアミノ酸残基に置換、挿入または欠失したタンパク質や、
（ＩＩＩ）配列番号１４に記載のアミノ酸配列のうち少なくとも１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を含むタンパク質や、
（ＩＶ）配列番号１４に記載のアミノ酸配列のうち少なくとも１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を含み、かつ前記アミノ酸残基のうちの一つ以上が他のアミノ酸残基に置換、挿入または欠失したタンパク質、
があげられる。
In this specification, the Fc-binding protein is an extracellular region of human FcγRI (specifically, in the case of natural human FcγRI, a region from the 16th glutamine to the 292nd histidine in the amino acid sequence shown in SEQ ID NO: 13). ) Or a protein constituting the extracellular region of human FcγRIIIa (specifically, in the case of natural human FcγRIIIa, the region from the 17th glycine to the 208th glutamine in the amino acid sequence shown in SEQ ID NO: 14) Say. However, it does not necessarily have to be the entire region of human FcγRI extracellular region or human FcγRIIIa extracellular region, and at least an antibody (immunoglobulin) among proteins (polypeptides) constituting human FcγRI extracellular region or human FcγRIIIa extracellular region The polypeptide of the area | region which can express the original function couple | bonded with Fc area | region of this should just be included. As an example of the Fc binding protein,
(I) a protein comprising an amino acid residue from at least the 16th glutamine to the 289th valine in the amino acid sequence of SEQ ID NO: 13,
(II) contains at least amino acid residues from the 16th glutamine to the 289th valine in the amino acid sequence shown in SEQ ID NO: 13, and at least one of the amino acid residues is substituted with another amino acid residue Inserted or deleted proteins,
(III) a protein comprising an amino acid residue from at least the 17th glycine to the 192nd glutamine in the amino acid sequence of SEQ ID NO: 14,
(IV) comprising at least the 17th glycine to the 192nd glutamine amino acid residue in the amino acid sequence of SEQ ID NO: 14, and at least one of the amino acid residues is substituted with another amino acid residue Inserted or deleted protein,
Can be given.
前記（ＩＩ）の具体例としては、特開２０１１−２０６０４６号公報や特開２０１４−０２７９１６号公報に開示のＦｃ結合性タンパク質があげられる。前記（ＩＶ）の具体例としては、配列番号１４に記載のアミノ酸配列のうち１７番目から１９２番目までのアミノ酸残基を含み、かつ当該１７番目から１９２番目までのアミノ酸残基において以下の（１）から（４０）のうち少なくともいずれか１つのアミノ酸置換が生じている、Ｆｃ結合性タンパク質（特願２０１３−２０２２４５号）があげられる。
（１）配列番号１４の１８番目のメチオニンがアルギニンに置換
（２）配列番号１４の２７番目のバリンがグルタミン酸に置換
（３）配列番号１４の２９番目のフェニルアラニンがロイシンまたはセリンに置換
（４）配列番号１４の３０番目のロイシンがグルタミンに置換
（５）配列番号１４の３５番目のチロシンがアスパラギン酸、グリシン、リジン、ロイシン、アスパラギン、プロリン、セリン、スレオニン、ヒスチジンのいずれかに置換
（６）配列番号１４の４６番目のリジンがイソロイシンまたはスレオニンに置換
（７）配列番号１４の４８番目のグルタミンがヒスチジンまたはロイシンに置換
（８）配列番号１４の５０番目のアラニンがヒスチジンに置換
（９）配列番号１４の５１番目のチロシンがアスパラギン酸またはヒスチジンに置換
（１０）配列番号１４の５４番目のグルタミン酸がアスパラギン酸またはグリシンに置換
（１１）配列番号１４の５６番目のアスパラギンがスレオニンに置換
（１２）配列番号１４の５９番目のグルタミンがアルギニンに置換
（１３）配列番号１４の６１番目のフェニルアラニンがチロシンに置換
（１４）配列番号１４の６４番目のグルタミン酸がアスパラギン酸に置換
（１５）配列番号１４の６５番目のセリンがアルギニンに置換
（１６）配列番号１４の７１番目のアラニンがアスパラギン酸に置換
（１７）配列番号１４の７５番目のフェニルアラニンがロイシン、セリン、チロシンのいずれかに置換
（１８）配列番号１４の７７番目のアスパラギン酸がアスパラギンに置換
（１９）配列番号１４の７８番目のアラニンがセリンに置換
（２０）配列番号１４の８２番目のアスパラギン酸がグルタミン酸またはバリンに置換
（２１）配列番号１４の９０番目のグルタミンがアルギニンに置換
（２２）配列番号１４の９２番目のアスパラギンがセリンに置換
（２３）配列番号１４の９３番目のロイシンがアルギニンまたはメチオニンに置換
（２４）配列番号１４の９５番目のスレオニンがアラニンまたはセリンに置換
（２５）配列番号１４の１１０番目のロイシンがグルタミンに置換
（２６）配列番号１４の１１５番目のアルギニンがグルタミンに置換
（２７）配列番号１４の１１６番目のトリプトファンがロイシンに置換
（２８）配列番号１４の１１８番目のフェニルアラニンがチロシンに置換
（２９）配列番号１４の１１９番目のリジンがグルタミン酸に置換
（３０）配列番号１４の１２０番目のグルタミン酸がバリンに置換
（３１）配列番号１４の１２１番目のグルタミン酸がアスパラギン酸またはグリシンに置換
（３２）配列番号１４の１５１番目のフェニルアラニンがセリンまたはチロシンに置換
（３３）配列番号１４の１５５番目のセリンがスレオニンに置換
（３４）配列番号１４の１６３番目のスレオニンがセリンに置換
（３５）配列番号１４の１６７番目のセリンがグリシンに置換
（３６）配列番号１４の１６９番目のセリンがグリシンに置換
（３７）配列番号１４の１７１番目のフェニルアラニンがチロシンに置換
（３８）配列番号１４の１８０番目のアスパラギンがリジン、セリン、イソロイシンのいずれかに置換
（３９）配列番号１４の１８５番目のスレオニンがセリンに置換
（４０）配列番号１４の１９２番目のグルタミンがリジンに置換
Specific examples of the (II) include Fc-binding proteins disclosed in JP2011-206046A and JP2014-027916A. Specific examples of the above (IV) include the amino acid residues from the 17th to the 192nd in the amino acid sequence shown in SEQ ID NO: 14, and the following (1 ) To (40), and Fc-binding protein (Japanese Patent Application No. 2013-202245) in which at least one amino acid substitution has occurred.
(1) 18th methionine of SEQ ID NO: 14 is replaced with arginine (2) 27th valine of SEQ ID NO: 14 is replaced with glutamic acid (3) 29th phenylalanine of SEQ ID NO: 14 is replaced with leucine or serine (4) 30th leucine of SEQ ID NO: 14 is replaced with glutamine (5) 35th tyrosine of SEQ ID NO: 14 is replaced with any of aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, and histidine (6) The 46th lysine of SEQ ID NO: 14 is replaced with isoleucine or threonine (7) The 48th glutamine of SEQ ID NO: 14 is replaced with histidine or leucine (8) The 50th alanine of SEQ ID NO: 14 is replaced with histidine (9) No. 14 51st tyrosine is aspartic acid or histidine (10) The 54th glutamic acid of SEQ ID NO: 14 is replaced with aspartic acid or glycine (11) The 56th asparagine of SEQ ID NO: 14 is replaced with threonine (12) The 59th glutamine of SEQ ID NO: 14 is replaced with arginine (13) 61st phenylalanine of SEQ ID NO: 14 is replaced with tyrosine (14) 64th glutamic acid of SEQ ID NO: 14 is replaced with aspartic acid (15) 65th serine of SEQ ID NO: 14 is replaced with arginine (16) The 71st alanine of No. 14 is substituted with aspartic acid (17) The 75th phenylalanine of SEQ ID No. 14 is substituted with leucine, serine or tyrosine (18) The 77th aspartic acid of SEQ ID No. 14 is substituted with asparagine (19) The 78th alanine of SEQ ID NO: 14 is changed to serine (20) The 82nd aspartic acid of SEQ ID NO: 14 is replaced with glutamic acid or valine (21) The 90th glutamine of SEQ ID NO: 14 is replaced with arginine (22) The 92nd asparagine of SEQ ID NO: 14 is replaced with serine ( 23) 93th leucine of SEQ ID NO: 14 is replaced with arginine or methionine (24) 95th threonine of SEQ ID NO: 14 is replaced with alanine or serine (25) 110th leucine of SEQ ID NO: 14 is replaced with glutamine (26 ) The 115th arginine of SEQ ID NO: 14 is replaced with glutamine (27) The 116th tryptophan of SEQ ID NO: 14 is replaced with leucine (28) The 118th phenylalanine of SEQ ID NO: 14 is replaced with tyrosine (29) 119th lysine substituted with glutamic acid (30) sequence The 120th glutamic acid of No. 14 is substituted with valine (31) The 121st glutamic acid of SEQ ID No. 14 is substituted with aspartic acid or glycine (32) The 151st phenylalanine of SEQ ID No. 14 is substituted with serine or tyrosine (33) The 155th serine of No. 14 is replaced with threonine (34) The 163rd threonine of SEQ ID NO: 14 is replaced with serine (35) The 167th serine of SEQ ID NO: 14 is replaced with glycine (36) The 169th position of SEQ ID NO: 14 (37) 171th phenylalanine of SEQ ID NO: 14 replaced with tyrosine (38) 180th asparagine of SEQ ID NO: 14 replaced with lysine, serine or isoleucine (39) of SEQ ID NO: 14 185th threonine is replaced by serine (40) 14 192 th glutamine substituted lysine
本発明は、以下の効果を奏することができる。
（１）本発明の水素酸化細菌は、目的タンパク質遺伝子および輸送タンパク質遺伝子（分泌性タンパク質遺伝子または膜タンパク質遺伝子）を導入し、前記目的タンパク質と前記輸送タンパク質とを共発現させることで、これまで困難であった目的タンパク質の培地中への分泌発現を可能とする。具体的には、本発明の水素酸化細菌は大腸菌よりも優れた分泌特性を有した細菌である。
（２）本発明の水素酸化細菌は安価なコストで目的タンパク質の生産が可能である。通常、組換え微生物で目的タンパク質を発現させるには、窒素源として酵母エキスやペプトン、炭素源としてグルコン酸やフルクトース、添加物としてアミノ酸やビタミン、といった高価な原料を用いる必要があった。一方本発明の水素酸化細菌を用いた目的タンパク質の分泌発現系では、窒素源としてアンモニア、アンモニウム塩、硝酸塩、亜硝酸塩などの無機窒素原料を用いることができ、炭素源として二酸化炭素ガス、炭酸塩など無機炭素原料を用いることができる。本発明の水素酸化細菌は、これらの無機窒素原料や無機炭素原料、および菌の生育に必要最低限の塩を含んだ培地原料のみから、目的タンパク質の分泌発現が可能である。
（３）本発明の水素酸化細菌を用いた目的タンパク質の製造方法は、従来の方法よりタンパク質精製のコストを抑えることができる。通常、組換え微生物で目的タンパク質の生産させるには、培養した微生物から目的タンパク質を抽出する操作が必要であり、かつ培地中の窒素源由来の不純物や前記微生物由来の夾雑タンパク質などにより精製が妨害されることから、精製コストが上昇する。一方本発明では、無機塩原料を用い、さらに目的タンパク質のみを培養液中に分泌発現させることが可能なため、菌体を除去するだけで高純度のタンパク質を得ることができ、精製工程を簡素化できる。
The present invention can achieve the following effects.
(1) The hydrogen-oxidizing bacterium of the present invention has been difficult until now by introducing a target protein gene and a transport protein gene (secretory protein gene or membrane protein gene) and co-expressing the target protein and the transport protein. It is possible to secrete and express the target protein in the medium. Specifically, the hydrogen-oxidizing bacterium of the present invention is a bacterium having secretion characteristics superior to that of E. coli.
(2) The hydrogen-oxidizing bacterium of the present invention can produce the target protein at a low cost. Usually, in order to express a target protein in a recombinant microorganism, it is necessary to use expensive raw materials such as yeast extract and peptone as a nitrogen source, gluconic acid and fructose as a carbon source, and amino acids and vitamins as additives. On the other hand, in the secretory expression system of the target protein using the hydrogen-oxidizing bacterium of the present invention, inorganic nitrogen materials such as ammonia, ammonium salt, nitrate, nitrite and the like can be used as the nitrogen source, and carbon dioxide gas and carbonate as the carbon source. An inorganic carbon raw material can be used. The hydrogen-oxidizing bacterium of the present invention is capable of secreting and expressing the target protein only from these inorganic nitrogen raw materials, inorganic carbon raw materials, and medium raw materials containing the minimum salt necessary for the growth of bacteria.
(3) The method for producing a target protein using the hydrogen-oxidizing bacterium of the present invention can reduce the cost of protein purification compared to the conventional method. Usually, in order to produce a target protein with a recombinant microorganism, it is necessary to extract the target protein from the cultured microorganism, and the purification is hindered by impurities from the nitrogen source in the culture medium or contaminating proteins derived from the microorganism. As a result, the purification cost increases. On the other hand, in the present invention, since the inorganic salt raw material can be used and only the target protein can be secreted and expressed in the culture solution, a high-purity protein can be obtained simply by removing the cells, and the purification process is simplified. Can be
以下、タンパク質としてＦｃ結合性タンパク質を、水素酸化細菌としてＲａｌｓｔｏｎｉａ属の菌を、それぞれ用いたときの実施例を用いて、本発明をさらに詳細に説明するが、本発明は前記例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples using Fc-binding proteins as proteins and Ralstonia bacteria as hydrogen-oxidizing bacteria. However, the present invention is limited to the above examples. It is not a thing.
実施例１ 発現ベクターの作製
下記（ａ）から（ｃ）に示すプラスミド（発現ベクター）を作製した。
Example 1 Preparation of Expression Vector Plasmids (expression vectors) shown in the following (a) to (c) were prepared.
（ａ）ファジンプロモーター、（Ｈ１６＿Ａ２９３５遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子、およびターミネーター遺伝子を導入したプラスミド
（ａ−１）ファジンプロモーター、（Ｈ１６＿Ａ２９３５遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子、ターミネーター遺伝子の順番に連結した遺伝子（配列番号１）をプラスミドｐＵＣ５７に挿入したものを、人工遺伝子合成により作製した（Ｇｅｎ Ｓｃｒｉｐｔ社 人工遺伝子合成受託サービス、ｐＵＣ５７はＧｅｎ Ｓｃｒｉｐｔ社提供の人工遺伝子挿入用標準ベクター）。なお配列番号１中、７番目から４４４番目の領域がファジンプロモーター（配列番号２）に、４５１番目から６８４番目の領域がＨ１６＿Ａ２９３５遺伝子（配列番号３）に、６９１番目から７９８番目までの領域が切断リンカー遺伝子（配列番号４）に、８０５番目から１６４１番目までがＦｃ結合性タンパク質遺伝子（配列番号５）に、１６４８番目から１７５８番目までの領域がターミネーター遺伝子（配列番号６）に、それぞれ相当する。またファジンプロモーターはＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株由来のプロモーターを、切断リンカーはＰａｕｃｉｍｏｎａｓ ｌｅｍｏｉｇｎｅｉの細胞外ＰＨＢ分解酵素のシグナル配列であるＰｒｅｐｈａＺ１を、それぞれ用い、Ｈ１６＿Ａ２９３５遺伝子、切断リンカー遺伝子およびＦｃ結合性タンパク質遺伝子はＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株におけるコドンに最適化したヌクレオチド配列となっている。以下、当該プラスミドをＡ２９３５−ＦｃＲ／ｐＵＣ５７と記載する。
（ａ−２）作製したＡ２９３５−ＦｃＲ／ｐＵＣ５７で大腸菌ＪＭ１０９株を形質転換した。得られた形質転換体を培養し、プラスミドＡ２９３５−ＦｃＲ／ｐＵＣ５７を抽出した。
（ａ−３）市販の広域宿主ベクターであるｐＢＢＲ１ＭＣＳ２で大腸菌ＪＭ１０９株を形質転換し、得られた形質転換体の培養物からプラスミド精製キットでｐＢＢＲ１ＭＣＳ２を調製した。
（ａ−４）得られたｐＢＢＲ１ＭＣＳ２をＡｐａＩとＸｂａＩで消化し、アガロース電気泳動後、約５．１ｋｂｐのＤＮＡ産物をゲル抽出キットにより精製した。
（ａ−５）（ａ−２）で調製したＡ２９３５−ＦｃＲ／ｐＵＣ５７をＡｐａＩとＸｂａＩで制限酵素消化し、アガロース電気泳動後、得られた約１．８ｋｂｐのＤＮＡ断片をゲル抽出キットにより精製した。
（ａ−６）（ａ−４）で得られたＤＮＡ断片と（ａ−５）で得られたＤＮＡ断片とを、１６℃でライゲーション反応（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ、東洋紡社製）を行ない、得られたプラスミドで大腸菌ＪＭ１０９株（タカラバイオ社製）を形質転換した。
（ａ−７）形質転換後の溶液を、カナマイシン２０μｇ／ｍＬとグルコース１％（ｗ／ｖ）とを含むＬＢ平板培地（バクトトリプトン１０ｇ／Ｌ、酵母エキス５ｇ／Ｌ、塩化ナトリウム１０ｇ／Ｌ、バクトアガロース１５ｇ／Ｌ）に撒き、目的クローンを選定した。
（ａ−８）選定した複数のコロニーを培養してプラスミド抽出を行ない、様々な制限酵素による切断パターンを確認した。
(A) Phasin promoter, (H16_A2935 gene-cleaved linker gene-Fc binding protein gene) fusion gene, and plasmid (a-1) introduced with terminator gene (H16_A2935 gene-cleaved linker gene-Fc binding property) A gene in which a gene (sequence number 1) linked in the order of a fusion gene of a protein gene and a terminator gene was inserted into a plasmid pUC57 was prepared by artificial gene synthesis (Gen Script, artificial gene synthesis contract service, pUC57 is Gen Script) Provided artificial gene insertion standard vector). In SEQ ID NO: 1, the 7th to 444th regions are cleaved by the phasin promoter (SEQ ID NO: 2), the 451th to 684th regions are the H16_A2935 gene (SEQ ID NO: 3), and the 691st to 798th regions are cleaved. In the linker gene (SEQ ID NO: 4), the region from 805 to 1641 corresponds to the Fc binding protein gene (SEQ ID NO: 5), and the region from 1648 to 1758 corresponds to the terminator gene (SEQ ID NO: 6). In addition, a promoter derived from Ralstonia eutropha H16 strain was used as the fasin promoter, PrephaZ1 which is a signal sequence of the extracellular PHB-degrading enzyme of Paucimonas lemignei was used as the cleavage linker, and the H16_A2935 gene, the cleavage linker gene and the Fc binding protein gene were used as Ralstonia eutropha The nucleotide sequence is optimized for codons in the H16 strain. Hereinafter, this plasmid is referred to as A2935-FcR / pUC57.
(A-2) Escherichia coli JM109 strain was transformed with the prepared A2935-FcR / pUC57. The obtained transformant was cultured, and plasmid A2935-FcR / pUC57 was extracted.
(A-3) Escherichia coli JM109 strain was transformed with pBBR1MCS2, which is a commercially available broad-area host vector, and pBBR1MCS2 was prepared from the resulting transformant culture with a plasmid purification kit.
(A-4) The obtained pBBR1MCS2 was digested with ApaI and XbaI, and after agarose electrophoresis, a DNA product of about 5.1 kbp was purified by a gel extraction kit.
(A-5) A2935-FcR / pUC57 prepared in (a-2) was digested with restriction enzymes with ApaI and XbaI, and after agarose electrophoresis, the obtained DNA fragment of about 1.8 kbp was purified with a gel extraction kit. .
(A-6) The DNA fragment obtained in (a-4) and the DNA fragment obtained in (a-5) were subjected to a ligation reaction (Ligation High, manufactured by Toyobo Co., Ltd.) at 16 ° C. to obtain Escherichia coli JM109 strain (Takara Bio Inc.) was transformed with the plasmid.
(A-7) An LB plate medium (bactotryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) containing 20 μg / mL kanamycin and 1% glucose (w / v) was added to the solution after transformation. , Bactoagarose 15 g / L), and the target clone was selected.
(A-8) A plurality of selected colonies were cultured, and plasmid extraction was performed to confirm cleavage patterns by various restriction enzymes.
以上の方法で、プラスミドＡ２９３５−ＦｃＲ／ｐＵＣ５７のＡｐａＩ／ＸｂａＩ断片をｐＢＢＲ１ＭＣＳ２のＡｐａＩ／ＸｂａＩ部位に挿入したプラスミドである、Ａ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２を得た。なおＡ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２は、ファジンプロモーター、（Ｈ１６＿Ａ２９３５遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子およびターミネーター遺伝子が、ｐＢＢＲ１ＭＣＳ２のＬａｃＺに対し、正方向に挿入されたプラスミドである。またＡ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２で大腸菌ＪＭ１０９に形質転換して得られた形質転換体を、以下、Ａ２９３５−ＦｃＲ／ＪＭ１０９とする。 By the above method, A2935-FcR / pBBR1MCS2, which is a plasmid in which the ApaI / XbaI fragment of plasmid A2935-FcR / pUC57 was inserted into the ApaI / XbaI site of pBBR1MCS2, was obtained. A2935-FcR / pBBR1MCS2 is a plasmid in which a fazine promoter, a fusion gene of (H16_A2935 gene-cleaved linker gene-Fc binding protein gene) and a terminator gene are inserted in the positive direction with respect to LacZ of pBBR1MCS2. The transformant obtained by transforming E. coli JM109 with A2935-FcR / pBBR1MCS2 is hereinafter referred to as A2935-FcR / JM109.
（ｂ）ファジンプロモーター、（Ｈ１６＿Ａ２８２０遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子およびターミネーター遺伝子を導入したプラスミド
（ｂ−１）Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株由来のＨ１６＿Ａ２８２０をＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株に適したコドンで変換して得られたヌクレオチド配列（配列番号１１の７番目から３３６番目の領域）を含むポリヌクレオチド（配列番号１１）をプラスミドｐＵＣ５７に挿入したプラスミドＡ２８２０／ｐＵＣ５７を人工遺伝子合成により作製した（Ｇｅｎ Ｓｃｒｉｐｔ社 人工遺伝子合成受託サービス）。
（ｂ−２）作製したＡ２８２０／ｐＵＣ５７で大腸菌ＪＭ１０９株を形質転換し、得られた形質転換体の培養物からプラスミドＡ２８２０／ｐＵＣ５７を抽出した。
（ｂ−３）（ａ）で作製したＡ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２をＨｉｎｄＩＩＩとＳｐｅＩで制限酵素消化し、アガロース電気泳動後、得られた約６．６ｋｂｐのＤＮＡ断片をゲル抽出キットにより精製した。
（ｂ−４）（ｂ−２）で調製したＡ２８２０／ｐＵＣ５７をＨｉｎｄＩＩＩとＳｐｅＩで制限酵素消化し、アガロース電気泳動後、得られた約０．３ｋｂｐのＤＮＡ断片をゲル抽出キットにより精製した。
（ｂ−５）（ｂ−３）で得られたＤＮＡ断片と（ｂ−４）で得られたＤＮＡ断片とを、１６℃でライゲーション反応（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ、東洋紡社製）を行ない、得られたプラスミドで大腸菌ＪＭ１０９株を形質転換した。
（ｂ−６）形質転換後の溶液を、カナマイシン２０μｇ／ｍＬとグルコース１％（ｗ／ｖ）とを含むＬＢ平板培地に撒き、目的クローンを選定した。
（ｂ−７）選定した複数のコロニーを培養してプラスミド抽出を行ない、様々な制限酵素による切断パターンを確認した。
(B) Phasin promoter, (H16_A2820 gene-cleaving linker gene-Fc binding protein gene) plasmid and terminator gene introduced plasmid (b-1) H16_A2820 derived from Ralstonia eutropha H16 strain is suitable for Ralstonia eutropha H16 strain Plasmid A2820 / pUC57, in which a polynucleotide (SEQ ID NO: 11) containing a nucleotide sequence obtained by codon conversion (region 7 to 336 of SEQ ID NO: 11) was inserted into plasmid pUC57, was prepared by artificial gene synthesis ( Gen Script artificial gene synthesis contract service).
(B-2) Escherichia coli JM109 strain was transformed with the prepared A2820 / pUC57, and plasmid A2820 / pUC57 was extracted from the culture of the obtained transformant.
(B-3) A2935-FcR / pBBR1MCS2 prepared in (a) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 6.6 kbp was purified by a gel extraction kit.
(B-4) A2820 / pUC57 prepared in (b-2) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 0.3 kbp was purified by a gel extraction kit.
(B-5) The DNA fragment obtained in (b-3) and the DNA fragment obtained in (b-4) were subjected to a ligation reaction (Ligation High, manufactured by Toyobo Co., Ltd.) at 16 ° C. to obtain Escherichia coli JM109 strain was transformed with the plasmid.
(B-6) The transformed solution was spread on an LB plate medium containing 20 μg / mL kanamycin and 1% (w / v) glucose, and the target clone was selected.
(B-7) A plurality of selected colonies were cultured and plasmid extraction was performed, and cleavage patterns with various restriction enzymes were confirmed.
以上の方法で、プラスミドＡ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２中のＨ１６＿Ａ２９３５遺伝子をＨ１６＿Ａ２８２０遺伝子に置き換えたプラスミドである、Ａ２８２０−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２を得た。なおＡ２８２０−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２は、ファジンプロモーター、（Ｈ１６＿Ａ２８２０遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子およびターミネーター遺伝子が、ｐＢＢＲ１ＭＣＳ２のＬａｃＺに対し、正方向に挿入されたプラスミドである。 By the above method, A2820-FcR / pBBR1MCS2, which is a plasmid in which the H16_A2935 gene in the plasmid A2935-FcR / pBBR1MCS2 is replaced with the H16_A2820 gene, was obtained. A2820-FcR / pBBR1MCS2 is a plasmid in which a fazine promoter, a fusion gene of (H16_A2820 gene-cleaved linker gene-Fc binding protein gene) and a terminator gene are inserted in the positive direction with respect to LacZ of pBBR1MCS2.
（ｃ）ファジンプロモーター、（Ｈ１６＿Ａ０９８３Ｎ末端領域遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子、およびターミネーター遺伝子を導入したプラスミド
（ｃ−１）Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株由来のＨ１６＿Ａ０９８３Ｎ末端領域を含む遺伝子産物をＰＣＲで増幅した。ＰＣＲ条件は、鋳型としてＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株のゲノムＤＮＡ（ＤＳＭＺ社製、ＤＳＭＺ４２８）を、プライマーセットとしてＨｉｎｄＩＩＩ部位を有した配列番号８に記載の配列からなるオリゴヌクレオチドとＳｐｅＩ部位を有した配列番号９に記載の配列からなるオリゴヌクレオチドとを、ポリメラーゼとしてＫＯＤ ＦＸ（東洋紡社製）を、それぞれ用い、９４℃で２分加熱後、９８℃で１０秒−６３℃で３０秒−６８℃で１分の温度サイクルを４０サイクル行ない、最後は６８℃で７分加熱する条件で行なった。
（ｃ−２）ＰＣＲ産物（配列番号１２）をアガロース電気泳動後、得られた約０．６ｋｂｐのＤＮＡ断片をゲル抽出キットにより精製した。
（ｃ−３）精製したＰＣＲ産物をリン酸化後、Ｍｉｇｈｔｙ Ｃｌｏｎｉｎｇ Ｋｉｔ（タカラバイオ社製）を用いてあらかじめＨｉｎｃＩＩで処理したｐＵＣ１１８とライゲーション反応を行ない、得られたプラスミドで大腸菌ＪＭ１０９株を形質転換した。
（ｃ−４）形質転換後の溶液を、カナマイシン１００μｇ／ｍＬを含むＬＢ平板培地に撒き、目的クローンを選定した。
（ｃ−５）選定した複数のコロニーを培養してプラスミド抽出を行ない、様々な制限酵素による切断パターンを確認した。得られたプラスミドＡ０９８３Ｎ／ｐＵＣ１１８は、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株ゲノム中のＨ１６＿Ａ０９８３のＮ末端側をコードする遺伝子（配列番号１２）をｐＵＣ１１８のＨｉｎｃＩＩ部位へ挿入したプラスミドである。
（ｃ−６）（ａ）で作製したＡ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２をＨｉｎｄＩＩＩとＳｐｅＩで制限酵素消化し、アガロース電気泳動後、得られた約６．６ｋｂｐのＤＮＡ断片をゲル抽出キットにより精製した。
（ｃ−７）（ｃ−５）で調製したＡ０９８３Ｎ／ｐＵＣ１１８をＨｉｎｄＩＩＩとＳｐｅＩで制限酵素消化し、アガロース電気泳動後、得られた約０．６ｋｂｐのＤＮＡ断片をゲル抽出キットにより精製した。
（ｃ−８）（ｃ−６）で得られたＤＮＡ断片と（ｃ−７）で得られたＤＮＡ断片とを、１６℃でライゲーション反応（Ｌｉｇａｔｉｏｎ Ｈｉｇｈ、東洋紡社製）を行ない、得られたプラスミドで大腸菌ＪＭ１０９株を形質転換した。
（ｃ−９）形質転換後の溶液を、カナマイシン２０μｇ／ｍＬとグルコース１％（ｗ／ｖ）とを含むＬＢ平板培地に撒き、目的クローンを選定した。
（ｃ−１０）選定した複数のコロニーを培養してプラスミド抽出を行ない、様々な制限酵素による切断パターンを確認した。
(C) Phasin promoter, (H16_A0983 N-terminal region gene-cleaved linker gene-Fc-binding protein gene) gene, and plasmid introduced with terminator gene (c-1) Gene containing H16_A0983 N-terminal region derived from Ralstonia eutropha H16 strain The product was amplified by PCR. PCR conditions were as follows: Ralstonia eutropha H16 strain genomic DNA (manufactured by DSMZ, DSMZ428) as a template, and a primer set having a HindIII site and an oligonucleotide consisting of the sequence described in SEQ ID NO: 8 and SEQ ID NO: 9 having a SpeI site Each of the oligonucleotides having the sequence described in 1 above was used as a polymerase using KOD FX (Toyobo Co., Ltd.), heated at 94 ° C. for 2 minutes, then 98 ° C. for 10 seconds to 63 ° C. for 30 seconds to 68 ° C. for 1 minute The temperature cycle was performed for 40 cycles, and finally the heating was performed at 68 ° C. for 7 minutes.
(C-2) After agarose electrophoresis of the PCR product (SEQ ID NO: 12), the obtained DNA fragment of about 0.6 kbp was purified by a gel extraction kit.
(C-3) After phosphorylation of the purified PCR product, ligation reaction was performed with pUC118 previously treated with HincII using the Mighty Cloning Kit (manufactured by Takara Bio Inc.), and Escherichia coli JM109 was transformed with the resulting plasmid. .
(C-4) The transformed solution was spread on an LB plate medium containing 100 μg / mL of kanamycin, and a target clone was selected.
(C-5) A plurality of selected colonies were cultured and plasmid extraction was performed, and cleavage patterns with various restriction enzymes were confirmed. The resulting plasmid A0983N / pUC118 is a plasmid in which the gene (SEQ ID NO: 12) encoding the N-terminal side of H16_A0983 in the Ralstonia eutropha H16 strain genome is inserted into the HincII site of pUC118.
(C-6) A2935-FcR / pBBR1MCS2 prepared in (a) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 6.6 kbp was purified by a gel extraction kit.
(C-7) A0983N / pUC118 prepared in (c-5) was digested with restriction enzymes HindIII and SpeI, and after agarose electrophoresis, the obtained DNA fragment of about 0.6 kbp was purified by a gel extraction kit.
(C-8) The DNA fragment obtained in (c-6) and the DNA fragment obtained in (c-7) were subjected to a ligation reaction (Ligation High, manufactured by Toyobo Co., Ltd.) at 16 ° C. to obtain Escherichia coli JM109 strain was transformed with the plasmid.
(C-9) The transformed solution was spread on an LB plate medium containing 20 μg / mL kanamycin and 1% glucose (w / v), and the target clone was selected.
(C-10) A plurality of selected colonies were cultured, and plasmid extraction was performed, and cleavage patterns with various restriction enzymes were confirmed.
以上の方法で、Ａ２９３５−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２中のＨ１６＿Ａ２９３５遺伝子を、Ｈ１６＿Ａ０９８３のＮ末端側をコードする遺伝子で置き換えたプラスミドであるＡ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２を得た。なおＡ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２は、ファジンプロモーター、（Ｈ１６＿Ａ０９８３遺伝子−切断リンカー遺伝子−Ｆｃ結合性タンパク質遺伝子）の融合遺伝子およびターミネーター遺伝子が、ｐＢＢＲ１ＭＣＳ２のＬａｃＺに対し、正方向に挿入されたプラスミドである。またＡ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２で大腸菌ＪＭ１０９株を形質転換して得られた形質転換体を、以下、Ａ０９８３Ｎ−ＦｃＲ／ＪＭ１０９とする。 By the above method, A0983N-FcR / pBBR1MCS2, which is a plasmid in which the H16_A2935 gene in A2935-FcR / pBBR1MCS2 was replaced with a gene encoding the N-terminal side of H16_A0983, was obtained. A0983N-FcR / pBBR1MCS2 is a plasmid in which a fazine promoter, a fusion gene of (H16_A0983 gene-cleavable linker gene-Fc binding protein gene) and a terminator gene are inserted in the positive direction with respect to LacZ of pBBR1MCS2. The transformant obtained by transforming E. coli JM109 strain with A0983N-FcR / pBBR1MCS2 is hereinafter referred to as A0983N-FcR / JM109.
なお、Ｆｃ結合性タンパク質遺伝子を含まないネガティブコントロールとして、ｐＢＢＲ１ＭＣＳ２で大腸菌ＪＭ１０９株を形質転換した株も作製した。この株を、以下、ＢＢＲ１ＭＣＳ２／ＪＭ１０９とする。 As a negative control not containing the Fc binding protein gene, a strain obtained by transforming E. coli JM109 strain with pBBR1MCS2 was also prepared. This strain is hereinafter referred to as BBR1MCS2 / JM109.
実施例２ Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ形質転換体の作製
以下の方法により、実施例１（ｂ）で作製したＡ２８２０−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２、実施例１（ｃ）で作製したＡ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２、またはｐＢＢＲ１ＭＣＳ２で、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａを形質転換した。
（１）Ａ２８２０−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２、Ａ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２またはｐＢＢＲ１ＭＣＳ２で、接合性大腸菌Ｓ１７−１を形質転換した。
（２）形質転換後の溶液を、カナマイシン５０μｇ／ｍＬを含むＬＢ平板培地に撒き、目的クローンを選定した。
（３）選定した大腸菌Ｓ１７−１株形質転換体を、カナマイシン５０μｇ／ｍＬを含むＬＢ（バクトトリプトン１０ｇ／Ｌ、酵母エキス５ｇ／Ｌ、塩化ナトリウム１０ｇ／Ｌ）培地中、３７℃で一晩培養を行なった。
（４）市販のＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株（ＡＴＣＣ １７６９９）またはａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ＿４株（ＤＳＭ ５４１）をそれぞれ抗生物質を含まないＮｕｔｒｉｅｎｔ Ｂｒｏｔｈ培地（Ｄｉｆｃｏ社製 Ｎｕｔｒｉｅｎｔ Ｂｒｏｔｈ、８ｇ／Ｌ）中で３０℃で一晩培養した。
（５）（４）で培養した菌体を遠心して濃縮し、６００ｎｍでの菌の濁度を測定後、（３）で培養した各大腸菌Ｓ１７−１株の形質転換体と菌体濁度１：１の比で混合し、抗生物質を含まないＮｕｔｒｉｅｎｔ ｂｒｏｔｈプレート（Ｎｕｔｒｉｅｎｔ Ｂｒｏｔｈ８ｇ／Ｌ、バクトアガロース１５ｇ／Ｌ）に撒き３０℃で一晩培養した。なおコントロールとして、各大腸菌Ｓ１７−１株の形質転換体、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株またはＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ＿４株のみを、抗生物質を含まないＮｕｔｒｉｅｎｔ ｂｒｏｔｈプレートで３０℃で一晩培養した。
（６）プレート表面の菌体をＮｕｔｒｉｅｎｔ Ｂｒｏｔｈ培地に懸濁させ、Ｎｕｔｒｉｅｎｔ Ｂｒｏｔｈ培地で１／１０００に希釈した後、カナマイシン６００μｇ／ｍＬを含むＮｕｔｒｉｅｎｔ ｂｒｏｔｈプレートに塗布し、３０℃で２日から３日培養した。コントロールである、各大腸菌Ｓ１７−１株の形質転換体、Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株またはＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ＿４株のみを培養したプレートからはコロニーは０または１個しか出現しなかった。一方、大腸菌Ｓ１７−１株の形質転換体とＲａｌｓｔｏｎｉａ属の株とを接合し得られた混合菌体を塗布したプレートからは、Ａ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２の大腸菌Ｓ１７−１形質転換体とＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株の混合物を撒いたプレートを除き、それぞれ数十個のコロニーが得られた。
（７）得られた接合菌体の各クローンを培養してプラスミド抽出を行ない、様々な制限酵素による切断パターンの分析により、目的のプラスミドが伝達されたことを確認した。
Example 2 Production of Ralstonia eutropha transformant A Ralstonia with A2820-FcR / pBBR1MCS2 produced in Example 1 (b), A0983N-FcR / pBBR1MCS2 or pBBR1MCS2 produced in Example 1 (c) by the following method. Eutropha was transformed.
(1) Conjugative E. coli S17-1 was transformed with A2820-FcR / pBBR1MCS2, A0983N-FcR / pBBR1MCS2, or pBBR1MCS2.
(2) The transformed solution was spread on an LB plate medium containing 50 μg / mL of kanamycin, and the target clone was selected.
(3) The selected Escherichia coli S17-1 strain transformant is overnight at 37 ° C. in LB (bactotryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) medium containing 50 μg / mL kanamycin. Culture was performed.
(4) Commercially available Ralstonia eutropha H16 strain (ATCC 17699) or alstonia eutropha PHB_4 strain (DSM 541) in Nutrient Broth medium (Difco's Nutrient Broth, 8 g / L) overnight without antibiotics Cultured.
(5) The cells cultured in (4) are centrifuged and concentrated. After measuring the turbidity of the cells at 600 nm, the transformants of each E. coli S17-1 strain cultured in (3) and the
(6) Cells on the surface of the plate are suspended in a nutrient broth medium, diluted to 1/1000 in a nutrient broth medium, and then applied to a nutrient broth plate containing 600 μg / mL kanamycin, and then at 30 ° C. for 2 to 3 days. Cultured. Only 0 or 1 colony appeared from a plate in which only the transformant of each Escherichia coli S17-1 strain, Ralstonia eutropha H16 strain or Ralstonia eutropha PHB_4 strain, was cultured as a control. On the other hand, from the plate coated with the mixed cell obtained by conjugating the transformant of E. coli S17-1 and the strain of the genus Ralstonia, the E. coli S17-1 transformant of A0983N-FcR / pBBR1MCS2 and Ralstonia eutropha H16 Dozens of colonies were obtained in each case, except for the plates plated with the strain mixture.
(7) Each clone of the obtained conjugated cells was cultured and plasmid extraction was performed, and it was confirmed that the target plasmid was transferred by analysis of cleavage patterns with various restriction enzymes.
以上の方法で、
Ａ２８２０−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２（実施例１（ｂ）で作製したプラスミド）をＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株に接合伝達した株であるＡ２８２０−ＦｃＲ／Ｈ１６株、
Ａ２８２０−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２（実施例１（ｂ）で作製したプラスミド）をＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ＿４株に接合伝達した株であるＡ２８２０−ＦｃＲ／ＰＨＢ＿４株、
Ａ０９８３Ｎ−ＦｃＲ／ｐＢＢＲ１ＭＣＳ２（実施例１（ｃ）で作製したプラスミド）をＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ＿４株に接合伝達した株であるＡ０９８３Ｎ−ＦｃＲ／ＰＨＢ＿４株、
ｐＢＢＲ１ＭＣＳ２（市販プラスミド）をＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ Ｈ１６株に接合伝達した株であるＢＢＲ１ＭＣＳ２／Ｈ１６株、および
ｐＢＢＲ１ＭＣＳ２（市販プラスミド）をＲａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ ＰＨＢ＿４株に接合伝達した株であるＢＢＲ１ＭＣＳ２／ＰＨＢ＿４株、
を得ることができた（以下、菌株を上記の名称で記載する）。
With the above method,
A2820-FcR / pBBR1MCS2 (plasmid prepared in Example 1 (b)) A2820-FcR / H16 strain, which is a strain obtained by conjugation to Ralstonia eutropha H16 strain,
A2820-FcR / PHB_4 strain, which is a strain obtained by conjugating A2820-FcR / pBBR1MCS2 (plasmid prepared in Example 1 (b)) to Ralstonia eutropha PHB_4,
A0983N-FcR / PHB_4 strain, which is a strain obtained by conjugating A0983N-FcR / pBBR1MCS2 (plasmid prepared in Example 1 (c)) to Ralstonia eutropha PHB_4,
BBR1MCS2 / H16 strain, which is a strain obtained by conjugating pBBR1MCS2 (commercial plasmid) to Ralstonia eutropha H16 strain, and BBR1MCS2 / PHB strain, which is a strain obtained by conjugating pBBR1MCS2 (commercial plasmid) to Ralstonia eutropha PHB_4 strain
(Hereinafter, the strain is described with the above name).
実施例３ フラスコ培養におけるＦｃ結合性タンパク質の生産性評価
実施例２で作製した水素酸化細菌を用いて、Ｆｃ結合性タンパク質の分泌発現性を評価した。
（１）実施例２で作製した、Ａ２８２０−ＦｃＲ／Ｈ１６株、Ａ２８２０−ＦｃＲ／ＰＨＢ＿４株、Ａ０９８３Ｎ−ＦｃＲ／ＰＨＢ＿４株、ＢＢＲ１ＭＣＳ２／Ｈ１６株、ＢＢＲ１ＭＣＳ２／ＰＨＢ＿４株、および比較対照用として実施例１で作製したＡ２８２０−ＦｃＲ／ＪＭ１０９株、Ａ０９８３Ｎ−ＦｃＲ／ＪＭ１０９株、ＢＢＲ１ＭＣＳ２／ＪＭ１０９株を、それぞれ２×ＹＴ（バクトトリプトン１６ｇ／Ｌ、酵母エキス１０ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ）培地を用いて試験管で前培養した（３０℃、１８０ｒｐｍ、一晩）。なおＡ２８２０−ＦｃＲ／Ｈ１６株、Ａ２８２０−ＦｃＲ／ＰＨＢ＿４株、Ａ０９８３Ｎ−ＦｃＲ／ＰＨＢ＿４株、ＢＢＲ１ＭＣＳ２／Ｈ１６株およびＢＢＲ１ＭＣＳ２／ＰＨＢ＿４株はカナマイシン３００μｇ／ｍＬを、Ａ２８２０−ＦｃＲ／ＪＭ１０９株、Ａ０９８３Ｎ−ＦｃＲ／ＪＭ１０９株およびＢＢＲ１ＭＣＳ２／ＪＭ１０９株はカナマイシン５０μｇ／ｍＬを、それぞれ添加して前培養した。
（２）２×ＹＴ培地２０ｍＬを入れた１００ｍＬ容フラスコに、（１）の前培養液を２００μＬ植菌し、３０℃、１３０ｒｐｍで４．５時間培養を行なった。なおＡ２８２０−ＦｃＲ／Ｈ１６株、Ａ２８２０−ＦｃＲ／ＰＨＢ＿４株、Ａ０９８３Ｎ−ＦｃＲ／ＰＨＢ＿４株、ＢＢＲ１ＭＣＳ２／Ｈ１６株およびＢＢＲ１ＭＣＳ２／ＰＨＢ＿４株はカナマイシン３００μｇ／ｍＬを、Ａ２８２０−ＦｃＲ／ＪＭ１０９株、Ａ０９８３Ｎ−ＦｃＲ／ＪＭ１０９株およびＢＢＲ１ＭＣＳ２／ＪＭ１０９株はカナマイシン５０μｇ／ｍＬを、それぞれ添加して培養した。
（３）ＩＰＴＧ（イソプロピル−β−チオガラクトピラノシド）を最終濃度０．５ｍＭとなるよう添加し、さらにＡ２８２０−ＦｃＲ／Ｈ１６株、Ａ２８２０−ＦｃＲ／ＰＨＢ＿４株、Ａ２８２０−ＦｃＲ／ＪＭ１０９株、Ａ０９８３Ｎ−ＦｃＲ／ＰＨＢ＿４株、Ａ０９８３Ｎ−ＦｃＲ／ＪＭ１０９株の培養液にはグルコン酸ナトリウムを最終濃度４％（ｗ／ｖ）となるよう添加して、３０℃で引き続き２日間培養した。
（４）菌体を１ｍＬずつマイクロチューブに採取し、１５０００ｒｐｍで１０分間遠心分離することで上清と菌体ペレットを分離した。上清は別のマイクロチューブにそれぞれ５００μＬ分取し、６０％（ｖ／ｖ）グリセロール水溶液を５００μＬ加えて保存した。一方上清を除いた菌体ペレットは、タンパク抽出試薬１ｍＬ（リゾチーム０．２ｍｇ／ｍＬ、エチレンジアミン四酢酸１ｍＭ、フッ化フェニルメチルスルホニル１ｍＭ、Ｂｅｎｚｏｎａｓｅ １２５Ｕ／ｍＬを含むＢｕｇＢｕｓｔｅｒ（Ｎｏｖａｇｅｎ社製）タンパク質抽出試薬）を加えることで菌体内のタンパク質を抽出した。
（５）上清中と菌体中のＦｃ結合性タンパク質量を以下に示すＥＬＩＳＡ法で測定した。
（５−１）９６穴のＥＬＩＳＡプレート（Ｎｕｎｃ社製）にガンマグロブリン製剤１０μｇ／ｍＬ（化学及血清療法研究所製）を各ウェルに１００μＬずつ添加し、４℃で１８時間静置することにより固定した。
（５−２）ＴＢＳ緩衝液（０．２％（ｗ／ｖ）Ｔｗｅｅｎ ２０、１５０ｍＭ ＮａＣｌを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．５））で洗浄後、１％ＢＳＡを含むＴｗｅｅｎ ２０を除いたＴＢＳ緩衝液（１５０ｍＭ ＮａＣｌを含む２０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．５））を２００μＬずつ添加し、４℃で１８時間以上静置することでブロッキング操作を施した。
（５−３）ＴＢＳ緩衝液で洗浄後、保存した培養上清または菌体抽出物の希釈系列を各ウェルに１００μＬ添加し、固定化したガンマグロブリンと反応させた（３０℃、１時間）。
（５−４）反応終了後、ＴＢＳ緩衝液で洗浄し、１次抗体ａｎｔｉ−ｈＦｃγＲ１／ＣＤ６４抗体（Ｒ＆Ｄ社製 ＭＡＢ１２５７１）０．１μＬ／ｍＬを各ウェルに１００μＬ添加して反応させた（３０℃、１時間）。
（５−５）反応終了後、ＴＢＳ緩衝液で洗浄し、２次抗体Ｇｏａｔ ａｎｔｉ−Ｍｏｕｓｅ ＩｇＧ−ｈ＋Ｉ ＨＲＰ抗体（ＢＥＴＨＹＬ社製 Ａ９０−２１６Ｐ）を各ウェルに１００μＬずつ添加し反応させた（３０℃、１時間）。
（５−６）反応終了後、ＴＢＳ緩衝液で洗浄し、ＴＭＢ Ｐｅｒｏｘｉｄａｓｅ Ｓｕｂｓｔｒａｔｅ（ＫＰＬ社製）を各ウェルに５０μＬずつ添加し発色反応させた。
（５−７）発色後、１Ｍリン酸水溶液を添加して反応の停止させ、４５０ｎｍの吸光度を測定した。
Example 3 Evaluation of Fc Binding Protein Productivity in Flask Culture Using the hydrogen-oxidizing bacteria prepared in Example 2, the secretion expression of Fc binding protein was evaluated.
(1) A2820-FcR / H16 strain, A2820-FcR / PHB_4 strain, A0983N-FcR / PHB_4 strain, BBR1MCS2 / H16 strain, BBR1MCS2 / PHB_4 strain prepared in Example 2 and Example 1 as a comparative control The prepared A2820-FcR / JM109 strain, A0983N-FcR / JM109 strain, and BBR1MCS2 / JM109 strain were respectively used in 2 × YT (bactotryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) medium. Pre-cultured in a test tube (30 ° C., 180 rpm, overnight). In addition, A2820-FcR / H16 strain, A2820-FcR / PHB_4 strain, A0983N-FcR / PHB_4 strain, BBR1MCS2 / H16 strain and BBR1MCS2 / PHB_4 strain are 300 kg / mL of kanamycin, A2820-FcR / JM109 strain, A0983N-FcR / JF The strain and the BBR1MCS2 / JM109 strain were each precultured by adding 50 μg / mL of kanamycin.
(2) In a 100 mL flask containing 20 mL of 2 × YT medium, 200 μL of the preculture solution of (1) was inoculated, and cultured at 30 ° C. and 130 rpm for 4.5 hours. In addition, A2820-FcR / H16 strain, A2820-FcR / PHB_4 strain, A0983N-FcR / PHB_4 strain, BBR1MCS2 / H16 strain and BBR1MCS2 / PHB_4 strain are 300 kg / mL of kanamycin, A2820-FcR / JM109 strain, A0983N-FcR / JF The strain and the BBR1MCS2 / JM109 strain were added with 50 μg / mL kanamycin and cultured.
(3) IPTG (isopropyl-β-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and the A2820-FcR / H16 strain, A2820-FcR / PHB — 4 strain, A2820-FcR / JM109 strain, A0983N -Sodium gluconate was added to the culture solution of FcR / PHB_4 strain and A0983N-FcR / JM109 strain to a final concentration of 4% (w / v), followed by culturing at 30 ° C. for 2 days.
(4) 1 mL of the cells were collected in a microtube and centrifuged at 15000 rpm for 10 minutes to separate the supernatant and the cell pellet. The supernatant was aliquoted in 500 μL in separate microtubes, and 500 μL of 60% (v / v) glycerol aqueous solution was added and stored. On the other hand, the bacterial cell pellet from which the supernatant was removed was a protein extraction reagent containing 1 mL of protein extraction reagent (lysozyme 0.2 mg / mL,
(5) The amount of Fc binding protein in the supernatant and the bacterial cells was measured by the ELISA method shown below.
(5-1) To a 96-well ELISA plate (manufactured by Nunc), add 10 μg / mL of gamma globulin preparation (manufactured by Chemo-Serum Therapy Laboratories) to each well and leave at 4 ° C. for 18 hours. Fixed.
(5-2) After washing with TBS buffer (0.2% (w / v)
(5-3) After washing with TBS buffer, 100 μL of the stored culture supernatant or dilution series of bacterial cell extract was added to each well and allowed to react with the immobilized gamma globulin (30 ° C., 1 hour).
(5-4) After completion of the reaction, the well was washed with a TBS buffer, and 0.1 μL / mL of the primary antibody anti-hFcγR1 / CD64 antibody (MAB12571 manufactured by R & D) was added to each well to react (30 ° C. 1 hour).
(5-5) After completion of the reaction, the plate was washed with TBS buffer, and 100 μL of the secondary antibody Goat anti-Mouse IgG-h + I HRP antibody (ATH-216P manufactured by BETHYL) was added to each well to react (30 ° C. 1 hour).
(5-6) After completion of the reaction, the plate was washed with TBS buffer, and 50 μL of TMB Peroxidase Substrate (manufactured by KPL) was added to each well for color development reaction.
(5-7) After color development, 1M phosphoric acid aqueous solution was added to stop the reaction, and the absorbance at 450 nm was measured.
結果を図１に示す。ネガティブコントロール（Ｆｃ結合性タンパク質遺伝子を導入しない組換え微生物）である、ＢＢＲ１ＭＣＳ２／ＪＭ１０９株、ＢＢＲ１ＭＣＳ２／Ｈ１６株およびＢＢＲ１ＭＣＳ２／ＰＨＢ＿４株は、いずれもＦｃ結合性タンパク質を生産しなかった。一方Ｆｃ結合性タンパク質遺伝子および輸送タンパク質遺伝子を水素酸化細菌に導入した菌株である、Ａ２８２０−ＦｃＲ／Ｈ１６株、Ａ２８２０−ＦｃＲ／ＰＨＢ＿４株、Ａ０９８３Ｎ−ＦｃＲ／ＰＨＢ＿４株は、Ｆｃ結合性タンパク質をそれぞれ５６ｍｇ／Ｌ、２８ｍｇ／Ｌ、３３ｍｇ／Ｌ生産し、そのうち培地中への分泌比率はそれぞれ６４％、８％、７％であった。なおＦｃ結合性タンパク質遺伝子およびタンパク質輸送遺伝子を大腸菌に導入した菌株である、Ａ２８２０−ＦｃＲ／ＪＭ１０９株、Ａ０９８３Ｎ−ＦｃＲ／ＪＭ１０９株のＦｃ結合性タンパク質の生産量は、それぞれ１３ｍｇ／Ｌ、８ｍｇ／Ｌであり、かつ培地中への分泌はほとんど認められなかった。 The results are shown in FIG. None of the BBR1MCS2 / JM109 strain, BBR1MCS2 / H16 strain, and BBR1MCS2 / PHB_4 strain, which are negative controls (recombinant microorganisms into which no Fc binding protein gene was introduced), produced Fc binding protein. On the other hand, A2820-FcR / H16 strain, A2820-FcR / PHB_4 strain, and A0983N-FcR / PHB_4 strain, which are strains in which an Fc binding protein gene and a transport protein gene are introduced into hydrogen-oxidizing bacteria, / L, 28 mg / L and 33 mg / L were produced, and the secretion ratios into the medium were 64%, 8% and 7%, respectively. The production amounts of Fc-binding proteins of the A2820-FcR / JM109 strain and A0983N-FcR / JM109 strain, which are strains in which the Fc-binding protein gene and the protein transport gene are introduced into Escherichia coli, are 13 mg / L and 8 mg / L, respectively. And almost no secretion into the medium was observed.
このことから、目的タンパク質遺伝子および輸送タンパク遺伝子を水素酸化細菌に導入して得られる本発明の水素酸化細菌を培養し、前記目的タンパク質と前記輸送タンパク質とを共発現させることで、目的タンパク質の生産性が向上しており、かつ培地中への分泌比率も向上することがわかる。 From this, the target protein gene and the transport protein gene are introduced into the hydrogen-oxidizing bacterium, the hydrogen-oxidizing bacterium of the present invention is cultured, and the target protein and the transport protein are co-expressed to produce the target protein. It can be seen that the properties are improved and the secretion ratio into the medium is also improved.
実施例４ 高密度培養におけるＦｃ結合性タンパク質の生産性評価
実施例３において、特にＦｃ結合性タンパク質の分泌発現に優れていたＡ２８２０−ＦｃＲ／Ｈ１６株について高密度培養を実施し、ヒトＦｃ結合性タンパク質の分泌生産性を評価した。
（１）カナマイシン３００μｇ／ｍＬを含む培地Ａ、培地Ｂまたは培地Ｃをフラスコに入れ、Ａ２８２０−ＦｃＲ／Ｈ１６株を接種後、３０℃で１６時間前培養した。
培地Ａ：０．５％（ｗ／ｖ）グルコン酸ナトリウムを含む４×ＹＴ（バクトトリプトン３２ｇ／Ｌ、酵母エキス２０ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ）培地
培地Ｂ：２％（ｗ／ｖ）グルコン酸ナトリウムを含むＭＳＭ培地（※１）
培地Ｃ：２％（ｗ／ｖ）フルクトースを含むＭＳＭ培地（※１）
（※１）ＭＳＭ培地の組成：Ｎａ２ＨＰＯ４ ３．６ｇ／Ｌ、ＫＨ２ＰＯ４ ４．７ｇ／Ｌ、（ＮＨ４）２ＳＯ４ ３．０ｇ／Ｌ、ＭｇＳＯ４・７Ｈ２Ｏ ２．２ｇ／Ｌ、Ｔｒａｃｅ ｅｌｅｍｅｎｔ ｓｏｌｕｔｉｏｎ（※２） １０ｍＬ／Ｌ
（※２）Ｔｒａｃｅ ｅｌｅｍｅｎｔ ｓｏｌｕｔｉｏｎの組成：ＦｅＳＯ４・７Ｈ２Ｏ ２８ｇ／Ｌ、ＺｎＳＯ４・７Ｈ２Ｏ ２．３ｇ／Ｌ、ＣｕＳＯ４・５Ｈ２Ｏ １．２ｇ／Ｌ、ＭｎＳＯ４・５Ｈ２Ｏ ０．５ｇ／Ｌ、ＣａＣｌ２・２Ｈ２Ｏ ２．０ｇ／Ｌ、Ｈ３ＢＯ３ ４０ｍｇ／Ｌ、（ＮＨ４）６Ｍｏ７Ｏ２４ ０．１２ｇ／Ｌ、ＣｏＣｌ２ ０．２ｇ／Ｌ、ＮｉＣｌ２・６Ｈ２Ｏ ２０ｍｇ／Ｌ、ＣｒＣｌ２ ６ｍｇ／Ｌ、３５％（ｗ／ｖ）ＨＣｌ １０ｍＬ／Ｌ
（２）１Ｌジャーに培地Ａ、培地Ｂまたは培地Ｃを４００ｍＬ入れ、（１）の前培養液２０ｍＬを接種後、培養を開始した。培養はＤＯ−ＳＴＡＴ法にて撹拌速度を制御し、１４％アンモニア水溶液と５０％リン酸水溶液を適宜流加することでｐＨを７．０に調整した。増殖開始後、以下に示す溶液を各培地に適宜流加することで、炭素源を補給した。
培地Ａ用流加液：ＭｇＳＯ４・７Ｈ２Ｏ ４２ｇ／Ｌ、Ｔｒａｃｅ ｅｌｅｍｅｎｔ ｓｏｌｕｔｉｏｎ（※２） １６７ｍＬ／Ｌ、３５％（ｗ／ｖ）ＨＣｌ ４１７μＬ／Ｌを含む３３％（ｗ／ｖ）グルコン酸ナトリウム水溶液
培地Ｂ用流加液：ＭｇＳＯ４・７Ｈ２Ｏ ４６ｇ／Ｌ、Ｔｒａｃｅ ｅｌｅｍｅｎｔ ｓｏｌｕｔｉｏｎ（※２） １８４ｍＬ／Ｌを含む４１％（ｗ／ｖ）グルコン酸水溶液
培地Ｃ用流加液：ＭｇＳＯ４・７Ｈ２Ｏ ４６ｇ／Ｌ、Ｔｒａｃｅ ｅｌｅｍｅｎｔ ｓｏｌｕｔｉｏｎ（※２） １８４ｍＬ／Ｌを含む３３％（ｗ／ｖ）フルクトース水溶液の
（３）（２）の培養液を経時的に採取し６００ｎｍの菌体濁度を測定した。また採取した培養液の一部は、実施例３（４）および（５）に記載の方法で培地中および菌体中のヒトＦｃ結合性タンパク質量を測定した。
Example 4 Fc-binding protein productivity evaluation in high-density culture In Example 3, A2820-FcR / H16 strain, which was particularly excellent in secretion expression of Fc-binding protein, was subjected to high-density culture, and human Fc binding property was observed. Protein secretion productivity was evaluated.
(1) Medium A, medium B or medium C containing 300 μg / mL of kanamycin was placed in a flask and inoculated with A2820-FcR / H16 strain, and then precultured at 30 ° C. for 16 hours.
Medium A: 4 × YT (bactotryptone 32 g / L, yeast extract 20 g / L, sodium chloride 5 g / L) containing 0.5% (w / v) sodium gluconate Medium B: 2% (w / v ) MSM medium containing sodium gluconate (* 1)
Medium C: MSM medium containing 2% (w / v) fructose (* 1)
(* 1) Composition of MSM medium: Na 2 HPO 4 3.6 g / L, KH 2 PO 4 4.7 g / L, (NH 4 ) 2 SO 4 3.0 g / L, MgSO 4 .7H 2 O 2g / L, Trace element solution (* 2) 10mL / L
(* 2) Trace element solution composition: FeSO 4 · 7H 2 O 28 g / L, ZnSO 4 · 7H 2 O 2.3 g / L, CuSO 4 · 5H 2 O 1.2 g / L, MnSO 4 · 5H 2 O 0.5 g / L, CaCl 2 · 2H 2 O 2.0 g / L, H 3 BO 3 40 mg / L, (NH 4 ) 6 Mo 7 O 24 0.12 g / L, CoCl 2 0.2 g / L, NiCl 2 · 6H 2 O 20mg / L ,
(2) 400 mL of medium A, medium B, or medium C was placed in a 1 L jar, and after inoculating 20 mL of the preculture solution of (1), culture was started. In culture, the stirring speed was controlled by the DO-STAT method, and the pH was adjusted to 7.0 by appropriately feeding a 14% aqueous ammonia solution and a 50% aqueous phosphoric acid solution. After the start of growth, a carbon source was replenished by appropriately feeding the following solutions to each medium.
Feed solution for medium A: MgSO 4 · 7H 2 O 42 g / L, Trace element solution (* 2) 167 mL / L, 35% (w / v) HCl 33% (w / v) gluconic acid 417 μL / L Feed solution for sodium aqueous medium B: MgSO 4 .7H 2 O 46 g / L, trace element solution (* 2) 41% (w / v) gluconic acid aqueous medium C containing 184 mL / L: MgSO 4・ The culture solution of (3) (2) in 33% (w / v) fructose aqueous solution containing 7H 2 O 46 g / L, Trace element solution (* 2) 184 mL / L was collected over time, and the turbidity at 600 nm The degree was measured. In addition, a part of the collected culture broth was measured for the amount of human Fc-binding protein in the medium and cells by the method described in Example 3 (4) and (5).
培養時間と菌体濁度との関係を図２に示す。また図２のうち、（１）から（６）の培養液の培地中および菌体中に含まれるヒトＦｃ結合性タンパク質量を測定した結果を図３に示す。窒素源としてタンパク質を用いても（培地Ａ）、アンモニウム塩またはアンモニアを用いても（培地Ｂおよび培地Ｃ）を用いても、Ｆｃ結合性タンパク質を培地中に分泌発現していることがわかる。このことから、目的タンパク質遺伝子および輸送タンパク遺伝子を水素酸化細菌に導入して得られる本発明の水素酸化細菌を培養し、前記目的タンパク質と前記輸送タンパク質とを共発現させる、本発明の目的タンパク質の製造方法は、無機塩と糖のみを窒素源／炭素源としても培養液中へ目的タンパク質を分泌発現できることがわかる。 The relationship between culture time and microbial turbidity is shown in FIG. Moreover, the result of having measured the amount of human Fc binding protein contained in the culture medium of (1) to (6) and a microbial cell among FIG. 2 is shown in FIG. It can be seen that the Fc-binding protein is secreted and expressed in the medium regardless of whether protein is used as the nitrogen source (medium A), ammonium salt or ammonia (medium B and medium C). From this, the target protein of the present invention is obtained by culturing the hydrogen-oxidized bacterium of the present invention obtained by introducing the target protein gene and the transport protein gene into the hydrogen-oxidized bacterium, and co-expressing the target protein and the transport protein. It can be seen that the production method can secrete and express the target protein into the culture solution using only the inorganic salt and sugar as the nitrogen source / carbon source.
Claims (5)
前記輸送タンパク質が配列番号７に記載の配列からなるポリペプチドであり、
前記目的タンパク質遺伝子および輸送タンパク質遺伝子が切断リンカーを介して連結した、水素酸化細菌。 A hydrogen-oxidizing bacterium capable of expressing the target protein obtained by introducing a target protein gene and a transport protein gene into a hydrogen-oxidizing bacterium of the genus Ralstonia ,
The transport protein is a polypeptide consisting of the sequence shown in SEQ ID NO: 7,
A hydrogen-oxidizing bacterium in which the target protein gene and the transport protein gene are linked via a cleavage linker .
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