JP5796147B2 - ポリペプチド多孔質体及びその製造方法 - Google Patents
ポリペプチド多孔質体及びその製造方法 Download PDFInfo
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- JP5796147B2 JP5796147B2 JP2015513726A JP2015513726A JP5796147B2 JP 5796147 B2 JP5796147 B2 JP 5796147B2 JP 2015513726 A JP2015513726 A JP 2015513726A JP 2015513726 A JP2015513726 A JP 2015513726A JP 5796147 B2 JP5796147 B2 JP 5796147B2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/0241—Containing particulates characterized by their shape and/or structure
- A61K8/0279—Porous; Hollow
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/42—Amides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
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Description
(A)ジメチルスルホキシド
(B)ジメチルスルホキシドに無機塩を加えたもの
(C)N,N−ジメチルホルムアミドに無機塩を加えたもの
前記溶液生成工程で生成した溶液を水溶性溶媒に置換することによりポリペプチドゲルを得る工程と、前記ポリペプチドゲルを乾燥する工程を含むことを特徴とする。なお、ここで言う「ポリペプチドを溶解用溶媒に溶解させる」とは、ポリペプチドを溶解用溶媒に完全に溶解させることと、ポリペプチドの微粒子を溶解用溶媒中に分散させることで、ポリペプチドの微粒子を溶解用溶媒に実質的に溶解させた形態と為すことの両方を含む。以下、同一の意味において使用する。
(A)ジメチルスルホキシド
(B)ジメチルスルホキシドに無機塩を加えたもの
(C)N,N−ジメチルホルムアミドに無機塩を加えたもの
前記溶液生成工程で生成した溶液を水溶性溶媒に置換することによりポリペプチドゲルを得る工程と、前記ポリペプチドゲルを乾燥する工程により得られる。前記溶液生成工程と前記溶媒を水溶性溶媒に置換する工程の間に、型枠に流し込み所定の形状に成形する工程を入れるか、あるいは前記溶媒を水溶性溶媒に置換する工程の後にカットすることにより所定の形状とすることができる。得られたポリペプチド多孔質の内部にはジメチルスルホキシド及びN,N−ジメチルホルムアミドからなる群から選ばれる少なくとも一つが存在していてもよい。前記溶解用溶媒の存在量は、特に限定されるものではなく、前記溶液生成工程で生成される溶液を前記水溶性溶媒に置換した後において意図せずに残存する程度の量である。
(1)溶媒残量測定
内部標準として1,2−ジクロロエタン−ギ酸溶液 濃度3,100ppm(0.00310mg/ml)を準備した。タンパク質溶液(10mlのギ酸に0.1gのポリペプチド多孔質体を溶解したもの)500μlと内部標準溶液500μlを混合した。さらに、H−NMR測定のためアセトニトリル重溶媒を同量程度加え約2倍に希釈し、H−NMR測定を行った(NMRの機種:JEOL社 JNM−ECX 100)。内部標準試料1,2−ジクロロエタンのH−NMR積分強度とDMSOのH−NMR積分強度を比較した。検量線の作成は3ppm〜3000ppmのDMSO−ギ酸溶液を作成し、上記プロトコルにしたがって、検量線を作成した。検量線との比較から、タンパク質溶液中のDMSO濃度を求めた。DMSO濃度測定は、JEOL社製、核磁気共鳴装置(NMR)を用いた。
(2)粘度
KEM社製、EMS装置を使用した。
1.ポリペプチドの準備
<ADF3Kai−Aの遺伝子の合成>
ニワオニグモの2つの主要なしおり糸タンパク質の一つであるADF3の部分的なアミノ酸配列をNCBIのウェブデータベース(NCBIアクセッション番号:AAC47010、GI:1263287)より取得し、同配列のN末端に開始コドンと、His10タグ及びHRV3Cプロテアーゼ(Human rhinovirus 3Cプロテアーゼ)認識サイトからなるアミノ酸配列(配列番号5)を付加し、該配列のN末端の1残基目から631残基目までのアミノ酸配列(配列番号1)からなるポリペプチド(ADF3Kai−A)をコードする遺伝子を合成した。その結果、配列番号6に示す塩基配列からなるADF3Kai−Aの遺伝子が導入されたpUC57ベクター(遺伝子の5’末端直上流にNde Iサイト、及び5’末端直下流にXba Iサイト有り)を取得した。その後、同遺伝子をNde I及びEcoR Iで制限酵素処理し、pET22b(+)発現ベクターに組み換え、ADF3Kai−Aの遺伝子が導入されたpET22b(+)ベクターを得た。
前記で得られたADF3Kai−Aの遺伝子配列を含むpET22b(+)発現ベクターを、大腸菌Rosetta(DE3)に形質転換した。得られたシングルコロニーを、アンピシリンを含む2mLのLB培地で15時間培養後、同培養液1.4mlを、アンピシリンを含む140mLのLB培地に添加し、37℃、200rpmの条件下で、培養液のOD600が3.5になるまで培養した。次に、OD600が3.5の培養液を、アンピシリンを含む7Lの2×YT培地に50%グルコース140mLと共に加え、OD600が4.0になるまでさらに培養した。その後、得られたOD600が4.0の培養液に、終濃度が0.5mMになるようにイソプロピル−β−チオガラクトピラノシド(IPTG)を添加してタンパク質発現を誘導した。IPTG添加後2時間経過した時点で、培養液を遠心分離し、菌体を回収した。IPTG添加前とIPTG添加後の培養液から調製したタンパク質溶液をポリアクリルアミドゲルに泳動させたところ、IPTG添加に依存して目的サイズ(約56.1kDa)のバンドが観察され、目的とするタンパク質(ADF3Kai−A)が発現していることを確認した。
(1)遠沈管(1000ml)にADF3Kai−Aのタンパク質を発現している大腸菌の菌体約50gと、緩衝液AI(20mM Tris−HCl、pH7.4)300mlを添加し、ミキサー(IKA社製「T18ベーシック ウルトラタラックス」、レベル2)で菌体を分散させた後、遠心分離機(クボタ社製の「Model 7000」)で遠心分離(11,000g、10分、室温)し、上清を捨てた。
(2)遠心分離で得られた沈殿物(菌体)に緩衝液AIを300mlと、0.1MのPMSF(イソプロパノールで溶解)を3ml添加し、前記IKA社製のミキサー(レベル2)で3分間分散させた。その後、高圧ホモジナイザー(GEA Niro Saovi社製の「Panda Plus 2000」)を用いて菌体を繰り返し3回破砕した。
(3)破砕された菌体に、3w/v%のSDSを含む緩衝液B(50mM TrisーHCL、100mM NaCl、pH7.0)300mLを加え、前記IKA社製のミキサー(レベル2)で良く分散させた後、シェイカー(タイテック社製、200rpm、37℃)で60分間攪拌した。その後、前記クボタ社製の遠心分離機で遠心分離(11,000g、30分、室温)し、上清を捨て、SDS洗浄顆粒(沈殿物)を得た。
(4)SDS洗浄顆粒を100mg/mLの濃度になるよう1Mの塩化リチウムを含むDMSO溶液で懸濁し、80℃で1時間熱処理した。その後、前記クボタ社製の遠心分離機で遠心分離(11,000g、30分、室温)し、上清を回収した。
(5)回収した上清に対して3倍量のエタノールを準備し、エタノールに回収した上清を加え、室温で1時間静置した。その後、前記クボタ社製の遠心分離機で遠心分離(11,000g、30分、室温)し、凝集タンパク質を回収した。次に純水を用いて凝集タンパク質を洗浄し、遠心分離により凝集タンパク質を回収するという工程を3回繰り返した後、凍結乾燥機で水分を除き、凍結乾燥粉末を回収した。得られた凍結乾燥粉末における目的タンパク質ADF3Kai−A(約56.1kDa)の精製度は、粉末のポリアクリルアミドゲル電気泳動(CBB染色)の結果をTotallab(nonlinear dynamics ltd.)を用いて画像解析することにより確認した。その結果、ADF3Kai−Aの精製度は約85%であった。
クモ糸タンパク質(ADF3Kai−A)の粉末0.8gをDMSO(1MのLiClを含む)20mlに入れ、80℃で30分間溶解させた。その後、ゴミと泡を取り除いた。溶液の溶液粘度は30.8cP(センチポアズ)であった。この溶液を透析チューブ(三光純薬株式会社セルロースチューブ36/32)に入れた。
前記透析チューブを3リットルの純水で満たされたビーカに入れた。その後3時間静置させた後、水を入れ替え、計6回この操作を行った。これにより、溶液中のクモ糸タンパク質が凝集して、ほぼ全てのDMSOが水に置換されたヒドロゲルが作成できた。得られたゲルの水分率は95.3質量%であった。
前記ヒドロゲルを凍結乾燥機(東京理化器械製の「FDUー1200」)により、14Pa、−45℃の条件で15時間凍結乾燥した。
(1)得られた多孔質体の溶媒残量は多孔質体100gに対して2.63gであった。
(2)得られた多孔質の見掛けの密度は0.077g/cm3であった。なお、見掛けの密度は公知の手法に従って求めた。以下に示す実施例2〜4の密度(見掛けの密度)も同様にして求めた。
(3)得られた多孔質体の水分最大吸収倍率は15.4倍であった。この水分吸収倍率は、水分を最大吸収させ、傾けても水が濡れてこない点とした。以下も同様である。
(4)得られた多孔質体は図1〜4に示すとおりである。
実施例1と同一のポリペプチドを用い、操作も同様にして次のゲルを作製した。濃度の薄いゲルとして、ポリペプチド粉末0.4gを用いて20mg/mlのドープ(DMSO+1MのLiCl)を準備し、ゲルを作製した。ドープ粘度は13.9cPであった。得られたゲルの水分率は98.8質量%であった。このゲルを実施例1と同じ条件で真空凍結乾燥した。得られた多孔質体の密度は0.020g/cm3であった。得られた多孔質体の水分最大吸収倍率は7.2倍であった。
実施例1と同一のポリペプチドを用い、操作も同様にして次のゲルを作製した。ポリペプチド粉末0.4gを用いて20mg/mlのドープ(塩なしのDMSO)を準備し、ゲルを作製した。得られたゲルの水分率は97.4質量%であった。このゲルを実施例1と同じ条件で真空凍結乾燥した。得られた多孔質体の密度は0.036g/cm3であった。得られた多孔質体の水分最大吸収倍率は6.1倍であった。
実施例1と同一のポリペプチドを用い、操作も同様にして次のゲルを作製した。ポリペプチド粉末0.4gを用いて20mg/mlのドープ(DMF+1MのLiCl)を準備し、ゲルを作製した。得られたゲルの水分率は98.2質量%であった。このゲルを実施例1と同じ条件で真空凍結乾燥した。得られた多孔質体の密度は0.039g/cm3であった。得られた多孔質体の水分最大吸収倍率は3.8倍であった。
配列番号6 塩基配列
Claims (7)
- クモ糸タンパク質に由来するポリペプチド多孔質体であって、前記ポリペプチドは非水溶性ポリペプチドを含み、見掛けの密度が0.1g/cm3以下であり、
前記ポリペプチド多孔質体にはジメチルスルホキシド及びN,N−ジメチルホルムアミドからなる群から選ばれる少なくとも一つが内部に存在していることを特徴とするポリペプチド多孔質体。 - 前記ポリペプチド多孔質体の見掛けの密度が0.01〜0.08g/cm3である請求項1に記載のポリペプチド多孔質体。
- 前記ポリペプチド多孔質体の水分吸収倍率が2〜20倍である請求項1又は2に記載のポリペプチド多孔質体。
- クモ糸タンパク質に由来するポリペプチドを、下記(A)〜(C)からなる群から選ばれる少なくとも一つの溶解用溶媒に溶解させてポリペプチドの溶液を得る溶液生成工程と、
(A)ジメチルスルホキシド
(B)ジメチルスルホキシドに無機塩を加えたもの
(C)N,N−ジメチルホルムアミドに無機塩を加えたもの
前記溶液生成工程で生成した溶液を水溶性溶媒に置換することによりポリペプチドゲルを得る工程と、
前記ポリペプチドゲルを乾燥する工程を含むことを特徴とするポリペプチド多孔質体の製造方法。 - 前記ポリペプチド多孔質体は、前記ポリペプチドの溶液を水に置換した水溶液を用いて生成する請求項4に記載のポリペプチド多孔質体の製造方法。
- 前記水溶性溶媒に置換する工程は、前記溶解用溶媒に溶解させたポリペプチドの溶液を透析膜内に入れ、水溶性溶媒中に浸漬し、水溶性溶媒を1回以上入れ替える請求項4又は5に記載のポリペプチド多孔質体の製造方法。
- 前記乾燥は真空凍結乾燥である請求項4〜6のいずれかに記載のポリペプチド多孔質体の製造方法。
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WO2021065794A1 (ja) | 2019-09-30 | 2021-04-08 | Spiber株式会社 | タンパク質成形体の製造方法 |
WO2021066078A1 (ja) | 2019-09-30 | 2021-04-08 | Spiber株式会社 | 筋組織再生剤 |
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WO2020067572A1 (ja) | 2018-09-28 | 2020-04-02 | Spiber株式会社 | タンパク質組成物の製造方法 |
WO2021065794A1 (ja) | 2019-09-30 | 2021-04-08 | Spiber株式会社 | タンパク質成形体の製造方法 |
WO2021066078A1 (ja) | 2019-09-30 | 2021-04-08 | Spiber株式会社 | 筋組織再生剤 |
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EP2990413A1 (en) | 2016-03-02 |
JPWO2014175178A1 (ja) | 2017-02-23 |
EP2990413A4 (en) | 2016-10-05 |
WO2014175178A1 (ja) | 2014-10-30 |
US10065997B2 (en) | 2018-09-04 |
CN104936978A (zh) | 2015-09-23 |
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EP2990413B1 (en) | 2019-05-29 |
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