JP6757408B2 - 金属内包かご状タンパク質の製造方法 - Google Patents
金属内包かご状タンパク質の製造方法 Download PDFInfo
- Publication number
- JP6757408B2 JP6757408B2 JP2018526038A JP2018526038A JP6757408B2 JP 6757408 B2 JP6757408 B2 JP 6757408B2 JP 2018526038 A JP2018526038 A JP 2018526038A JP 2018526038 A JP2018526038 A JP 2018526038A JP 6757408 B2 JP6757408 B2 JP 6757408B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- metal
- cage protein
- encapsulated
- ferritin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Description
特開2012−200242号公報の実施例1に記載の方法に準じて、プラスミドpKL223を作製した。特開2008−194815号公報に記載のウマ由来フェリチン「CNHB−Fer0」のアミノ酸配列をコードする遺伝子を合成し(配列番号1)、開始コドンのすぐ上流にEcoRI及びWSD配列(GAATTCAGGAGGTATTAT、配列番号2)、終始コドンの下流にPstI配列(CTGCAG)を付加した。得られたDNA断片を、EcoRI及びPstIで消化し、プラスミドpKL223のtacプロモーターの下流に位置するEcoRI−PstIギャップに挿入して、プラスミドpKLCNH−Fer0を構築した。作製されたプラスミドpKLCNH−Fer0を大腸菌BL21に導入して、形質転換された大腸菌BL21(pKLCNH−Fer0)を得た。
本実施例では、すべての成分を混合した時点での最終組成が、
80mM HEPES(pH7.5)
0.5mg/mL アポフェリチン
5mM 硫酸アンモニウム鉄
20%(w/v) 二糖類(実施例1:スクロース、実施例2:トレハロース、実施例3:マルトース)
となるように、以下の手順にしたがって合計80mLの溶液を調製し、鉄をアポフェリチンに導入した。
以下では、便宜上スクロースを添加した実施例1の方法について述べるが、実施例2及び3では、スクロースに代えてトレハロース、マルトースをそれぞれ用いたこと以外は実施例1と同様の方法で行い、比較例1では、二糖類を添加しなかったこと以外は実施例1と同様の方法で行った。
カラム:Agilent Bio SEC−5 500A
カラム温度:25℃
移動相:50mM Tris−HCl緩衝液(pH8.0)
流速:1.0mL/分
注入量:50μL
検出:280nm
モノマー保持時間:8.9分付近
鉄の導入率は、上記鉄内包フェリチンを2mM Tris−HCl緩衝液(pH8.0)で5.0mg/mLとなるように調整し、更に50mM Tris−HCl緩衝液(pH8.0)で0.1mg/mLに希釈し、380nmにおける希釈液の吸光度を、分光光度計(NanoDrop ND−1000(Thermo Fisher Scientific社製))を用いて測定することで評価した。
また、ポジティブコントロールとして、特開2008−194815号公報の実施例(準備4−2、5及び6)に記載の方法に準じて鉄内包フェリチンを製造し、上記と同様の方法で鉄の導入率評価を行った。
結果を表1に示す。
上記実施例1において、スクロースに代えて以下の表2に記載の各多糖類を用いたこと以外は、上記実施例1と同様の方法でアポフェリチンへの鉄への導入を行い、鉄の導入率を評価した(実施例4〜10)。また、上記実施例1において、糖類を添加しなかったこと以外は、上記実施例1と同様の方法でアポフェリチンへの鉄への導入を行い、鉄の導入率を評価した(比較例2)。結果を表2に示す。
Claims (7)
- 多糖類の存在下で、かご状タンパク質に金属元素を導入して、前記金属元素を内包する金属内包かご状タンパク質を生成させる工程を備える、金属内包かご状タンパク質の製造方法であって、
前記かご状タンパク質がフェリチンであり、
前記金属元素が鉄であり、
前記金属内包かご状タンパク質が鉄内包フェリチンである、方法。 - 前記金属内包かご状タンパク質を生成させる工程の後に、前記金属内包かご状タンパク質を含む混合物をゲル濾過クロマトグラフィーで精製し、得られた溶出液をそのまま濃縮する工程を備える、請求項1に記載の製造方法。
- 前記多糖類がオリゴ糖である、請求項1又は2に記載の製造方法。
- 前記オリゴ糖が二糖類である、請求項3に記載の製造方法。
- 前記二糖類がスクロース、トレハロース及びマルトースからなる群から選択される少なくとも一種である、請求項4に記載の製造方法。
- 前記オリゴ糖が三糖類、四糖類又は五糖類である、請求項3に記載の製造方法。
- 前記多糖類がデキストリンである、請求項1又は2に記載の製造方法。
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