JP6853801B2 - 荷電栄養タンパク質および方法 - Google Patents
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Description
本出願は、2012年3月26日に出願された米国仮特許出願第61/615,816号に対する優先権を主張するものであり、参照により、その全体が本明細書に組み込まれる。
本出願は、EFS−Webを介してASCII形式で提出された配列表を含み、参照により、その全体が本明細書に組み込まれる。2013年3月12日に作成された上記ASCIIコピーは、1005.004−PCT_SL.txtという名称であり、1,194,666バイトのサイズである。
食事性タンパク質は、ヒトの健康および成長のための必須栄養素である。世界保健機関は、エネルギーバランスがとれている場合および体重が安定している場合、食事性タンパク質は、エネルギー摂取量の約10〜15%に寄与するべきであると推奨している。様々な国々におけるタンパク質の1日平均摂取量は、これらの推奨が世界中で消費されているタンパク質の量と一致することを示唆している。エネルギーバランスがとれた状態で摂取される場合、タンパク質からのエネルギーの平均20〜30%を含む食事が、高タンパク食の代表的なものである。
本開示の目的のために、「栄養タンパク質」は、望ましい量の必須アミノ酸を含有するタンパク質である。いくつかの実施形態において、栄養タンパク質は、少なくとも30重量%の必須アミノ酸を含む。いくつかの実施形態において、栄養タンパク質は、少なくとも40重量%の必須アミノ酸を含む。いくつかの実施形態において、栄養タンパク質は、少なくとも50重量%の必須アミノ酸を含む。いくつかの実施形態において、栄養タンパク質は、食用種において自然に生じるタンパク質またはタンパク質の断片を含むかまたはそれらからなる。その最も広義において、「食用種」は、少なくとも1種類の哺乳動物によって有害な影響なく食されることが分かっている任意の種を包含する。有害な影響は、毒性効果および毒作用を含む。いくつかの実施形態において、食用種は、有害な影響なくヒトによって食されることが分かっている種である。限定された地理的地域において、ある種類の哺乳動物の小さなグループのみの食事の、希少ではあるが既知である構成要素である食用種もあれば、世界中ほとんどの地域で主食となっている食用種もある。他の実施形態において、食用種は、いずれかの哺乳動物によって以前に食されたことは分かっていないが、試験時に食用であることが明らかになる種である。食用種は、限定されないが、以下を含むワタ(turneri)、シロノタモギタケモドキ、グリシンマックス、アジアイネ、メバチ、ブリストルコーンモミ、トゲネズミ、タクヨウレンリソウ、インドヤギュウ、ケープグリスボック、タテゴトアザラシ、カラチョウザメ、ジャワジャコウネコ、ヒマラヤヒラタケ、ダッタンソバ、ストローブマツ、アサガオ、キャラボク、フウリンアサガオ、オオノガイ、キウイフルーツ、グラントガゼル、ヨーロッパヤマナラシ、セイヨウスモモ、セグロカモメ、ビロードクサフジ、ホシエビス、ヒロハノマンテマ、サラワクイルカ、アメリカウバガイ、クシマンセ、アズキ、ツルナシレリンソウ、カラフトマス、ミシシッピワニ、アレッポマツ、カモメ、セイヨウアブラナ、シラタマソウ、クラカケアザラシ、インディアンガゼル、テーダマツ、カナダイチイ、ヒロハザミア、ウンナンマツ、ヒマラヤゴヨウマツ、アスパラガス、アヒ・アマリージョ、イガゴヨウマツ、ヨーロッパイチイ、シベリアマツ、オレンジ、ハワイアンスコーレルフィッシュ、アメリカバイソン、トムソンガゼル、ヤハズエンドウ、カナダガン、セロリ、コブカエデ、コリアンダー、ヒメシラタマソウ、レタス、カプシカム・シネンゼ、シラビソ、カプラ・ヒルクス、スペックガゼル、サケ、ヒメノアサガオ、シロウリ、ワモンアザラシ、アカギツネ、ルコウソウ、ソラナム(habrochaites)、ポプラ種、リギダマツ、オーバーカップオーク、ベニバナインゲン、ユリカモメ、トガリエビス、タイセイヨウクロマグロ、ホッキョクギツネ、インディアンバイソン、イタリアン・メープル、イロハモミジ、セイヨウヒイラギガシ、モンタナマツ、オオヅル、カギミヤママツ、ウメ、マスノスケ、コウジョウセンガゼル、フェネック、シシマツ、ワタ(barbadense)、シカモアカエデ、ベニザケ、ヒゲイノシシ、ソバ亜種アンセストラル、カルドン、ヤエナリ、セイコウハコヤナギ、ワタ(schwendimanii)、ソラナム(chacoense)、アカガシワ、キュウリ、サバンナシマウマ、ギンザケ、ラジアータパイン、ゼニガタアザラシ、オグロヅル、アメリカオオモミ、サクラマス、ホウレンソウ、ソラナム(chilense)、アダックス、サツマイモ、グレビーシマウマ、トドマツ、イタリアカサマツ、オオカグラコウモリ、オータム・クロッカス、ブンタン、シロチョウザメ、ワタ(gossypioides)、インドジャコウネコ、ターキー・オーク、インドガン、フォックステールパイン、スパニッシュムシトリナデシコ、オンコリンクス属種、ビルマジャコウネコ、ヤク、スラッシュマツ、インドノロバ、アイベックス、ニンニク、ラディッシュ、エキナタマツ、ブラックチェリー、ニジエビス、シロバナマンテマ、ヤセイカンラン、ノラニンジン、ニジマス、カイラン、ワタ(hirsutum)、ヨーロッパモミ、シトラス・レティキュラータ、チコリー、コープレイ、ラマ、トウモロコシ、セキショウ、キットギツネ、アルタイアルガリ、シャープグリスボック、コントルタマツ、コブウシ、シベリアアイベックス、ポンデローサマツ、アーモンド、ソラナム(sogarandinum)、ヨウサイ、ハナジロカマイルカ、ビッグホーン、セイヨウミザクラ、ダマガゼル、ビンナガ、マンテマ、スイスマツ、サフラン、スイカ、アカガゼル、ハリゲナタネ、マーコール、ミンドロスイギュウ、ダイオウマツ、セイヨウバクチノキ、マナヅル、マルバアサガオ、チワワマツ、ハラジロカマイルカ、スタインボック、ブラッシカ・ラパ亜種ペキネンシス、アメリカセンニチコウ、ホシアサガオ、パツラマツ、マスクメロン、バージニアマツ、ソラナム(lycopersicum)、アカマツ、アパッチパイン、ヨーロッパナラ、イポメア・セトーサ、トキイロヒラタケ、ハチマキカグラコウモリ、ヒツジ、ヒメエビス、ブロッコリー、カフカスツール、キタカミハクヨウ、プレーリーオニオン、ソラマメ、フツウソバ、ステップバイソン、コルクガシ、ラゴフィラ・ラモシッシマ、マダガスカルボア、シベリアチョウザメ、カプシクム・アンヌウム、パンコムギ、アフリカツメガエル、バイカルアザラシ、アドリアチョウザメ、オニマタタビ、ドールシープ、ソラナム(tuberosum)、カラバオ、ザボン、ヨーロッパバイソン、ヌビアノロバ、アノア、エリンギ、ソラナム(demissum)、ウリアル、トウモロコシ亜種パルビグルミス、ハットクマメ、ウェルウィッチア、オーストラリアヅル、マルバルコウ、タマネギ、ゼメリングガゼル、ブラッシカ・ラパ、ビクーニャ、ソラナム(peruvianum)、アフリカツメガエル、カフカスアイベックス、キハダマグロ、ヤマシマウマ、セキショクヤケイ、ソラナム(bulbocastanum)、テラソカグラコウモリ、タイセイヨウカマイルカ、カバ、チョウセンゴヨウ、フランスモミジ、アメリカクロポプラ、ブラック・コットンウッド、ロシアチョウザメ、クロマツ、キャベツ、カジカ(korotneffi)、エドミガゼル、ウラジロモミ、マンシュウモミ、マウンテンガゼル、ゴヨウマツ、ハボタン、ペポカボチャ、カザンマツ、オオシラビソ、タイセイヨウクロマグロ、ウンシュウミカン、ソラナム(cheesmanii)、カマイルカ、ノルウェーカエデ、レモン、デュメリルボア、ソラナム(commersonii)、ワタ(arboreum)、モモ、ヒラタケ、モミ、リムガゼル、タイセイヨウサケ、アメリカウミザリガニ、カリフォルニアアカモミ、バンテン、ゴマフアザラシ、フィリピンヒゲイノシシ、ナス、ゼニガタアザラシ、ヨーロッパアカマツ、ザミア、コサックギツネ、リーキ、カスピカイアザラシ、ケープギツネ、チュウゴクイチイ、カリフラワー、キバシハイイロガン、ライマメ、ブラッシカ・カンペストリス、サトウカエデ、ハイマツ、ソラナム(pennellii)、ピニョンマツ、イモネノホシアサガオ、ウラジロハコヤナギ、ノドキリマス、フユナラ、スンダイボイノシシ、プルツワルスキーモウコノウマ、コトカケヤナギ、アフリカツメガエル、タイヘイヨウイチイ、グアナコ、バンクスマツ、イヌホオズキ、セレベスイノブタ、カラシナ、ダンダラカマイルカ、アメリカヤマナラシ、コロラドトウヒ、ヤマアノア、カシ(gamelliflora)、アジアムフロン、アジアスイギュウ、リュウキュウマツ、フィリピンヒゲイノシシ、インゲンマメ、ブラウントラウト、ペルシャチョウザメ、ソラナム(brevidens)、レジノーサマツ、セーブルアンテロープ、ヌビアアイベックス、シーアスパラガス、イポメア・プラテンシス、ブタ、パサン、ガラスマメ、アオスジエビス、タイセイヨウオヒョウ、アメリカショウブ、ウマ、コブウシ、ツケバナ、アルパカ、フランスカイガンショウ、マダコ、ソラナム(crispum)、ローンアンテロープ、チャップマンシマウマ、アレキサンダークシマンセ、ヨルガオ、ヒトツブコムギ、アラビアバルサムノキ、ミナミカマイルカ、ドルカスガゼル、ケルメスオーク、ハクガン、トウ、ヒマラヤマツ、マンシュウクロマツ、フィッシェリ、ホオカザリヅル、カイロトゲマウス、ネットメロン、セイロンヤケイ、ピスム・サティヴム、ノブコーンパイン、サンドパイン、サウジガゼル、キャプラアイベックス、イポメア・トリフィーダ、テオシント、ベトナムマツ、ウイルソントゲマウス、パセリ、ピンオーク、コムギ(timopheevi)、シチメンチョウ、ヤセイカンラン、ヤセイカンラン、ビーツ、ソラナム(lycopersicum)、インゲンマメ、メカジキ、ストライプドバス、オオクチバス、マゼランツキヒガイ、ヨーロピアン・スプラト、タイセイヨウニシン、モトカタクチイワシ、セイヨウカボチャ、アガリクス・ビスポルス、キングバナナ、マルス・ドメスティカ、ヒヨコマメ、マガモ、ヒメツルコケモモ、ラズベリー、ローブッシュ・ブルーベリー、イチゴ、セイヨウヤブイチゴ、マスクメロン、パイナップル、ペポカボチャ、二ホンカボチャ、ブタ、バジル、ローズマリー、ウイキョウ、ダイオウ、パパイヤ、マンゴー、キウイフルーツ、アンズ、セイヨウミザクラ、ココヤシ、オリーブ、セイヨウナシ、イチジク、クダモノトケイソウ、アジアイネ亜種ジャポニカ、アジアイネ亜種インディカ、ヨーロッパウズラ、サッカロマイセス・セレビシエ。
電荷P=−0.002−(C)(0.045)−(D)(0.999)−(E)(0.998)+(H)(0.091)+(K)(1.0)+(R)(1.0)−(Y)(−0.001)
式中、Cは、ポリペプチド中のシステイン残基の数であり、Dは、アスパラギン酸残基の数であり、Eは、グルタミン酸残基の数であり、Hは、ヒスチジン残基の数であり、Kは、リジン残基の数であり、Rは、アルギニン残基の数であり、Yは、チロシン残基の数である。ポリペプチドのアミノ酸当たりの電荷(電荷A)は、正味電荷(電荷P)をアミノ酸残基の数(N)で除すことによって算出することができ、すなわち、電荷A=電荷P/Nである。(Bassi S(2007),“A Primer on Python for Life Science Researchers.”PLoS Comput Biol 3(11):e199.doi:10.1371/journal.pcbi.0030199を参照のこと)。
栄養ポリペプチドまたはタンパク質をコードする核酸も、本明細書において提供される。いくつかの実施形態において、核酸は、単離される。いくつかの実施形態において、核酸は、精製される。
本明細書においてさらに記載されるように、本明細書に開示される核酸分子のうちの少なくとも1つを含む発現ベクターを含むベクターもまた提供される。いくつかの実施形態において、ベクターは、本明細書に開示されるような栄養タンパク質をコードする少なくとも1つの単離された核酸分子を含む。代替の実施形態において、ベクターは、1つ以上の発現制御配列に動作可能に連結されたそのような核酸分子を含む。ベクターは、したがって、組換え微生物宿主細胞において少なくとも1つの組換えタンパク質を発現させるために使用することができる。
本明細書に記載される組換え遺伝子を発現させるために有用なプロモーターは、構成的プロモーターおよび誘導性/抑制可能プロモーターの両方を含む。誘導性/抑制可能プロモーターの例として、ニッケル誘導性プロモーター(例えば、PnrsA、PnrsB:例えば、Lopez−Mauy et al.,Cell(2002)v.43:247−256を参照)およびPnirA等の尿素抑制性プロモーター(例えば、Qi et al.,Applied and Environmental Microbiology(2005)v.71:5678−5684に記載される)が挙げられる。誘導性/抑制可能プロモーターのさらなる例として、PnirA(硝酸によって誘導され、尿素によって抑制される、nirA遺伝子の発現を駆動するプロモーター、)およびPsuf(鉄ストレスによって誘導される、sufB遺伝子の発現を駆動するプロモーター)が挙げられる。構成的プロモーターの例として、Pcpc(cpcオペロンの発現を駆動するプロモーター)、Prbc(ルビスコの発現を駆動するプロモーター)、PpsbAII(PpsbAIIの発現を駆動するプロモーター)、Pcro(croの発現を駆動するλファージプロモーター)が挙げられる。他の実施形態において、PaphIlおよび/またはlaclq−Ptrcプロモーターは、発現を制御するために使用することができる。遺伝子操作された微生物において複数の組換え遺伝子が発現される場合、異なる遺伝子が、異なるプロモーターによってもしくは別個のオペロンの同一のプロモーターによって制御されてもよいか、または2つ以上の遺伝子の発現が、オペロンの一部としての単一のプロモーターによって制御されてもよい。
また、本明細書に開示される核酸分子またはベクターで形質転換される宿主細胞、およびその子孫も提供される。いくつかの実施形態において、宿主細胞は、微生物細胞である。いくつかの実施形態において、宿主細胞は、自由に複製するベクターであってもよいが、そうである必要はないベクター上に、核酸配列を担持する。他の実施形態において、核酸は、宿主細胞のゲノムおよび/または宿主細胞の内在性プラスミドに組み込まれている。形質転換された宿主細胞は、例えば、本明細書に開示される組換え栄養タンパク質の生成に用途を見出す。
当業者は、本明細書に開示されるような組換え栄養タンパク質を生成する(および任意選択的に分泌する)ように組換え細胞を培養するための、ならびに発現させた組換えタンパク質の精製および/または単離のための多くの好適な方法を認識している。タンパク質精製のために選択される方法は、該当するタンパク質の特性、細胞内におけるその位置および形態、ベクター、宿主株のバックグラウンド、ならびに発現させたタンパク質の意図する用途を含む多くの変数に依存する。培養条件も、所与の標的タンパク質の溶解性および局在性に影響を及ぼし得る。本明細書に開示されるような組換え微生物細胞に発現される標的タンパク質を精製するために、限定されないが、イオン交換およびゲル濾過を含む多くのアプローチを用いることができる。
本開示の栄養タンパク質をコードする核酸配列を含む核酸分子は、核酸配列を含むトランスジェニック植物の生成を可能にする。したがって、本開示はまた、本開示の栄養タンパク質をコードする核酸配列を含む組換え核酸分子を含む植物も提供する。植物は、形質転換および再生を受けるいずれの植物であってもよく、限定されないが、アカシア、アルファルファ、アネット、リンゴ、アンズ、アーティチョーク、ルッコラ、アスパラガス、アボカド、バナナ、オオムギ、マメ、ビート、ブラックベリー、ブルーベリー、ブロッコリー、芽キャベツ、キャベツ、キャノーラ、カンタロープメロン、ニンジン、キャッサバ、カリフラワー、セロリ、白菜、サクランボ、コリアンダー、柑橘類、クレメンタイン、コーヒー、トウモロコシ、ワタ、キュウリ、ダグラスファー、ナス、エンダイブ、キクヂシャ、ユーカリ、ウイキョウ、イチジク、森林樹、ウリ、ブドウ、グレープフルーツ、ハニーデューメロン、ヒカマ、キウイフルーツ、レタス、リーク、レモン、ライム、テーダマツ、マンゴ、メロン、キノコ、木の実、オーツ麦、オクラ、タマネギ、オレンジ、観賞植物、パパイヤ、パセリ、エンドウ、モモ、ピーナッツ、ナシ、コショウ、カキ、パイナップル、オオバコ、プラム、ザクロ、ポプラ、ジャガイモ、カボチャ、マルメロ、ラジエータパイン、赤チコリ、ラディッシュ、ナタネ、ラズベリー、米、ライ麦、モロコシ、サザンパイン、大豆、ホウレンソウ、スカッシュ、イチゴ、テンサイ、サトウキビ、ヒマワリ、スイートコーン、サツマイモ、モミジバフウ、タンジェリン、紅茶、タバコ、トマト、芝生、つる植物、スイカ、コムギ、ヤムイモ、およびズッキーニを含む。好ましい実施形態において、植物は、マメ、ブロッコリー、キャベツ、キャノーラ、ニンジン、カリフラワー、セロリ、白菜、トウモロコシ、ワタ、キュウリ、ナス、リーク、レタス、メロン、エンドウ、コショウ、カボチャ、ラディッシュ、ホウレンソウ、大豆、スカッシュ、サトウキビ、スイートコーン、トマト、スイカ、およびコムギ植物である。いくつかの実施形態において、植物は、トウモロコシ植物である。いくつかの実施形態において、植物は、大豆植物である。いくつかの実施形態において、植物は、ワタ植物である。いくつかの実施形態において、植物は、キャノーラ植物である。いくつかの実施形態において、植物は、アラビドプシス、ベータ、グリシンヤトロファ、ミスカンサス、パニクム、ファラリス、ポプルス、サッカルム、サリクス、シモンドシア、およびゼアから選択される属のメンバーである。
本明細書に開示される少なくとも1つの栄養タンパク質は、栄養組成物を形成するための少なくとも1つの第2の構成成分と組み合わせられてもよい。いくつかの実施形態において、組成物中のアミノ酸の唯一の源は、本明細書に開示される少なくとも1つの栄養タンパク質である。そのような実施形態において、上記組成のアミノ酸組成物は、本明細書に開示される少なくとも1つの栄養タンパク質のアミノ酸組成物と同じである。いくつかの実施形態において、組成物は、本明細書に開示される少なくとも1つの栄養タンパク質と、少なくとも1つの第2のタンパク質とを含む。いくつかの実施形態において、少なくとも1つの第2のタンパク質は、本明細書に開示される第2の栄養タンパク質であり、他の実施形態において、少なくとも1つの第2のタンパク質は、本明細書に開示される栄養タンパク質ではない。いくつかの実施形態において、組成物は、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個、またはそれ以上の本明細書に開示される栄養タンパク質を含む。いくつかの実施形態において、組成物は0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個、またはそれ以上の本明細書に開示される栄養タンパク質ではないタンパク質を含む。いくつかの実施形態において、組成物は、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個、またはそれ以上の栄養タンパク質を含み、組成物は、0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個、またはそれ以上の本明細書に開示される栄養タンパク質ではないタンパク質を含む。
いくつかの実施形態において、本明細書に開示される栄養タンパク質および栄養組成物は、患者またはユーザ(時には、集合的に「対象」と称される)に投与される。本明細書において使用される場合、「投与する」および「投与」は、ある人物が別の人物に、特定の様式で、かつ/または特定の目的のために栄養タンパク質または栄養組成物を摂取するように指示する実施形態、同様に、ユーザが、第2の人物から受けたいずれの指示にも関係なくまたは矛盾して、特定の様式で、かつ/または特定の目的のために栄養タンパク質または栄養組成物を使用する状況を包含する。ある人物が別の人物に、特定の様式で、かつ/または特定の目的のために、栄養タンパク質または栄養組成物を摂取するように指示する実施形態の非限定的な例として、医師が一連の処置法および/または治療を患者に処方する場合、訓練者がユーザ(運動選手等)に、特定の一連の処置法および/または治療に従うように助言する場合、ならびに、製造業者、流通業者、マーケティング業者が、例えば、製品の販売またはマーケティングに関連して提供される、パッケージまたは他の材料上の広告または標示を通して、エンドユーザに使用の条件を推奨する場合が挙げられる。
UniProtKB/Swiss−Prot(European Bioinformatics InstituteとSwiss Institute of Bioinformaticsとのコラボレーション)は、手作業で処理および検討されたタンパク質のデータベースであり、タンパク質同定の出発点として使用した。50個以上のアミノ酸を有する食用種であるソラナム・リコペルシク、トウモロコシ、オリザ・サティバ亜種ジャポニカ、グリシンマックス、オービス・アリエス、ピスム・サティヴム、ホウレンソウ、オリザ・サティバ亜種インディカ、トリティクム・アエスティウム、スース・スクロファ、モモ、カプシクム・アンヌウム、マルス・ドメスティカ、キハダマグロ、カプラ・ヒルクス、シセル・アリエチヌム、タイセイヨウサケ、メレアグリス・ガロパボ、ソラナム・ツベロスム、およびアガリクス・ビスポルスからのタンパク質試料をこの試験の標的として採取した。こうして、8,415個の一連のタンパク質が評価のために提供された。各タンパク質について、アミノ酸含有量、必須アミノ酸(「EAA」)のパーセンテージ、分枝鎖アミノ酸(「BCAA」)のパーセンテージ、ロイシン(「L」)のパーセンテージ、およびタンパク質がすべての必須アミノ酸を含有するかどうかを算出した。また、SolvScoreも各タンパク質ごとに算出した。さらに、既知のアレルゲンに対して50%を超える全体的相同性を有するものがあるかどうかを決定するために、既知のアレルゲンのデータベースに対してタンパク質をスクリーニングした。全部で463個のタンパク質が、20以下のSolveScoreを有し、19%以上のEAA、8%以上のBCAA、および4%以上のLeuを含有し、既知のアレルゲンに対する50%未満の全体的相同性(配列番号1〜463)を有すると同定された。(同定されたタンパク質がゼラチンよりも高い含有量のEAA、BCAA、およびLeuを有することを確実にするために、これらの値は、この実施例において該当する栄養タンパク質を同定するために用いられた)。一連のタンパク質について、pH7での溶媒和スコア(「SolvScore」)、凝集スコアpH7での(「AggScore」)、アレルゲン性(すなわち、本明細書に記載されるような既知のアレルゲンに対する局所的相同性パーセント)、毒性(すなわち、本明細書に記載されるような既知の毒素に対する相同性パーセント)、抗栄養性(すなわち、本明細書に記載されるような既知のプロテアーゼ阻害剤に対する相同性パーセント)、およびヒト相同性(すなわち、本明細書に記載されるような既知のヒトタンパク質に対する相同性パーセント)を算出し、Cys残基(「C」)の合計数を決定した。このようにして同定した200個の代表的なタンパク質の特性を表3Aおよび3Bに提示する。
本開示の栄養タンパク質をコードする遺伝子を、大腸菌における発現のためにコドン最適化し、LifeTechnologies/GeneArtまたはDNA2.0のいずれかにより合成した。遺伝子は、天然タンパク質を発現するように、または精製を促進するために2つのアミノ末端タグのうちの1つを含有するように設計した:
本開示に記載されるような栄養タンパク質を大量に生成するための代表的なプロトコルは次の通りである。
一連の292個の栄養タンパク質をコードするオープンリーディングフレームをクローニングし、大腸菌に導入し、実施例2の方法を用いて組換えタンパク質の発現を評価した。用いたシステムにおいて、163個のタンパク質が発現されたと同定され、129個は発現されなかった。発現された163個のタンパク質のうち、125個を可溶性発現について調べた。75個は可溶性に発現されたが、50個は発現されなかったことが分かった。
実施例2および3に記載されるように生成した9個の栄養タンパク質の溶解性を、遠心濃縮後にタンパク質濃縮アッセイにより調べた。Coomassie Plus(Bradford)Protein Assay(Thermo Scientific)のプロトコルに従って、280nmの吸光度(適用される場合)で、20mM HEPES(pH7.5)中の試料をタンパク質濃度について検査した。これらの測定値に基づいて、10mgのタンパク質をAmicon Ultra 3kDa遠心濾過器(Millipore)に添加した。10,000Xgで30分間の遠心分離により試料を濃縮した。最終的な濃縮された試料を、沈殿したタンパク質および色について調べ、次いで、上記のようにタンパク質の濃度について検査した。その結果を表5に示す。
栄養タンパク質の熱安定性は、タンパク質が有用な有効期間を有する可能性があるかどうかの洞察を提供する。実施例6および7に記載されるように生成したタンパク質の試料を、迅速な熱安定性スクリーニング法を用いて並行してスクリーニングした。この方法では、温度の上昇に伴ってタンパク質が変性するにつれて形成する凝集タンパク質に結合する疎水性色素(Enzo Life Sciences,ProteoStat(登録商標)Thermal shift stability assay kit)の存在下で、タンパク質を2つの代表的な配合物中で25℃から95℃に徐々に加熱した(Niesen,F.H.,Berglund,H.&Vadadi,M.,2007.The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.Nature Protocols,Volume 2,pp.2212−2221)。結合すると色素の蛍光が著しく増加するので、次いでそれをrtPCR機器により記録し、タンパク質の溶融曲線によって表す(Lavinder,J.J.,Hari,S.B.,Suillivan,B.J.&Magilery,T.J.,2009.High−Throughput Thermal Scanning:A General,Rapid Dye−Binding Thermal Shift Screen for Protein Engineering.Journal of the American Chemical Society,pp.3794−3795.)。熱シフトが終了した後、試料を不溶性沈殿物について調べ、分析用サイズ排除クロマトグラフィー(SEC)によってさらに分析した。
タンパク質の消化率をスクリーニングする目的は、潜在的に安全ではないアレルゲン性タンパク質を除外すること、およびペプチドの生物学的利用能の予測因子として消化の相対的完全性を決定することである。このスクリーニング方法は、模擬胃内消化および模擬腸内消化といったタンパク質消化の両方の段階を含む、生理的に適切なインビトロ消化反応を利用する(Moreno,J.F.et al.,2005.Stability of the major allergen Brazil nut 2S albumin(Ber e 1)to physiologically relevant in vitro gastrointestinal digestion.FEBS Journal,pp.341−352)。試料は、反応を通して採取することができ、チップ電気泳動およびLC−QTOF−MSを用いてインタクトなタンパク質およびペプチド断片について分析することができる。アレルゲン性を有するタンパク質は、消化プロテアーゼに耐性を示すためにアレルギー反応を引き起こすより高いリスクを有するタンパク質またはタンパク質の大きな断片を同定することにより評価することができる(Goodman,R.E.et al.,2008.Allergenicity assessment of genetically modified crops−what makes sense?.Nature Biotechnology,pp.73−81)。消化率は、いかに効率的にタンパク質がペプチドに分解されるかを判定することによって測定される(Daniel,H.,2003.Molecular and Integrative Physiology of Intestinal Peptide Transport.Annual Review of Physiology,Volume 66,pp.361−384)。
実施例2および3に記載されるように生成した2つの栄養タンパク質(配列番号762および763)を、実施例7に記載されるようにSGFおよびSIFによる消化に供した。両方のタンパク質はSGF中で完全に消化され、SGF半減期を表8に示す。
Claims (42)
- 配列番号486で示されるポリペプチド配列に少なくとも90%相同なポリペプチド配列を含む組換え栄養タンパク質と賦形剤とを含む栄養組成物であって、組換え栄養タンパク質が少なくとも約1gの量で存在する栄養組成物。
- 組換え栄養タンパク質が異種微生物で生産される、請求項1に記載の栄養組成物。
- 賦形剤が、緩衝剤、保存剤、安定剤、結合剤、圧縮剤、滑沢剤、分散促進剤、崩壊剤、香味剤、甘味剤、及び着色剤からなる群から選択される、請求項1に記載の栄養組成物。
- 組換え栄養タンパク質が配列番号486で示されるポリペプチド配列に少なくとも90%相同なポリペプチド配列からなる、請求項1に記載の栄養組成物。
- 異種微生物が、Escherichia coli、Bacillus subtilis、Bacillus megaterium、Streptomyces coelicolo、Aspergillus niger、Aspergillus nidulans、Aspergillus oryzae、Trichoderma reesei、Saccharomyces cerevisiae、及びPichia stipitisからなる群から選択される、請求項2に記載の栄養組成物。
- 異種微生物が、Escherichia coli、Bacillus subtilis、Saccharomyces cerevisiae、Pichia pastoris、及びAspergillus nigerからなる群から選択される、請求項5に記載の栄養組成物。
- 異種微生物が、Bacillus subtilisである、請求項6に記載の栄養組成物。
- 栄養組成物が、液体溶液、スラリー、懸濁液、ゲル、ペースト、粉末、又は固体である、請求項1に記載の栄養組成物。
- 栄養組成物が、液体溶液である、請求項8に記載の栄養組成物。
- 栄養組成物が、経口経路のために製剤化されている、請求項1に記載の栄養組成物。
- 栄養タンパク質が、1g〜5g、2g〜10g、5g〜15g、10g〜20g、15g〜25g、20g〜40g、25〜50g、又は30〜60gから選択される量で存在する、請求項1に記載の栄養組成物。
- 栄養タンパク質が、15g〜25g、25〜50g、又は30〜60gから選択される量で存在する、請求項11に記載の栄養組成物。
- 栄養タンパク質が、少なくとも2g、3g、4g、5g、6g、7g、8g、9g、10g、15g、20g、25g、30g、35g、40g、45g、50g、55g、60g、65g、70g、75g、80g、85g、90g、95g、又は100gの量で存在する、請求項1に記載の栄養組成物。
- 栄養タンパク質が、少なくとも15gの量で存在する、請求項13に記載の栄養組成物。
- ポリペプチド配列が、pH7で少なくとも50g/Lの水可溶性を有する、請求項1に記載の栄養組成物。
- ポリペプチド配列が、pH7で少なくとも100g/Lの水可溶性を有する、請求項15に記載の栄養組成物。
- ポリペプチド配列が、30分よりも短い模擬胃内消化半減期を有する、請求項1に記載の栄養組成物。
- ポリペプチド配列が、10分よりも短い模擬胃内消化半減期を有する、請求項17に記載の栄養組成物。
- ポリペプチド配列が、2分よりも短い模擬胃内消化半減期を有する、請求項18に記載の栄養組成物。
- ポリペプチド配列が、模擬胃液内で30分内に完全に消化される、請求項17に記載の栄養組成物。
- 少なくとも1つの補助的なミネラル又はミネラル源を含む、請求項1に記載の栄養組成物。
- 補助的なミネラル又はミネラル源が、カルシウムである、請求項21に記載の栄養組成物。
- ポリペプチド配列が、pH7で12.5mg/mlの濃度で存在する際に、水溶液中、95℃で不溶性沈殿物を形成しない、請求項1に記載の栄養組成物。
- 賦形剤が、組成物中のアミノ酸の総重量の約50%以下、約45%以下、約40%以下、約35%以下、約30%以下、約25%以下、約20%以下、約15%以下、約10%以下、約5%以下、約2%以下、または約1%以下である、請求項1に記載の栄養組成物。
- ポリペプチド配列が、pH7で少なくとも50g/Lの水可溶性;10分よりも短い模擬胃内消化半減期を有し、ポリペプチド配列が、pH7で12.5mg/mlの濃度で存在する際に、水溶液中、95℃で不溶性沈殿物を形成しない、請求項1に記載の栄養組成物。
- ポリペプチド配列が、配列番号486で示されるポリペプチド配列に少なくとも95%相同であり;ポリペプチド配列が、2分よりも短い模擬胃内消化半減期を有する、請求項25に記載の栄養組成物。
- 組換え栄養タンパク質が、少なくとも15g〜25g、25〜50g、又は30〜60gの量で存在し、かつ栄養組成物が液体溶液である、請求項25に記載の栄養組成物。
- カルシウムを含む、請求項27に記載の栄養組成物。
- 賦形剤が、緩衝剤、保存剤、安定剤、分散促進剤、香味剤、甘味剤、及び着色剤からなる群から選択される、請求項3に記載の栄養組成物。
- 請求項28に記載の栄養組成物を含む、サルコペニア、悪液質、又は虚弱を治療するための医薬であって、栄養組成物が、ヒト対象に経口投与される医薬。
- 請求項28に記載の栄養組成物を含む、対象における筋肉量、筋力、及び筋機能的能力のうちの少なくとも1つを維持するか又は増加させるための組成物であって、栄養組成物が、ヒト対象に経口投与される組成物。
- 請求項1に記載の栄養組成物の少なくとも1の用量を含む、サルコペニア、悪液質、又は虚弱を治療するための医薬であって、栄養組成物の少なくとも1の用量が、少なくとも1日毎に少なくとも1日ヒト対象に投与され、かつ栄養タンパク質が、1g〜5g、2g〜10g、5g〜15g、10g〜20g、15g〜25g、20g〜40g、25〜50g、又は30〜60gから選択される量で存在する、医薬。
- 栄養タンパク質が、1日当たり1.5g/体重1kgの対象のタンパク質摂取量の少なくとも10%である、請求項32に記載の医薬。
- 栄養タンパク質が、少なくとも1つの薬学的又は生物学的薬物製品と同時に、ヒト対象に経口で摂取される、請求項32に記載の医薬。
- 請求項1に記載の栄養組成物の少なくとも1の用量を含む、望ましいボディマス指数を達成するための組成物であって、栄養組成物の少なくとも1の用量が、少なくとも毎日少なくとも1日ヒト対象に投与され、かつ栄養タンパク質が、1g〜5g、2g〜10g、5g〜15g、10g〜20g、15g〜25g、20g〜40g、25〜50g、又は30〜60gから選択される量で存在する、組成物。
- 請求項1に記載の栄養組成物の十分な量を含む、対象における筋肉量、筋力、及び筋機能的能力のうちの少なくとも1つを維持するか又は増加させるための組成物であって、栄養組成物の十分な量が対象に供される組成物。
- 栄養組成物が満腹感を誘導するのに有効な量で対象に供される、請求項35に記載の組成物。
- 栄養組成物が運動の遂行(performance)に合わせて対象によって摂取される、請求項32に記載の医薬又は請求項36に記載の組成物。
- 栄養組成物が経口又は経腸で対象によって摂取される、請求項32に記載の医薬又は請求項36に記載の組成物。
- 栄養組成物が経口で対象によって摂取される、請求項39に記載の医薬又は組成物。
- 栄養組成物が1日0.1g〜1g、1日1g〜5g、1日2g〜10g、1日5g〜15g、1日10g〜20g、1日15g〜30g、1日20g〜40g、1日25g〜50g、1日40g〜80g、又は1日50g〜100gの割合で摂取される、請求項32に記載の医薬又は請求項36に記載の組成物。
- 請求項27に記載の栄養組成物を含む、望ましいボディマス指数を達成するための組成物であって、栄養組成物がヒト対象に経口で投与される、組成物。
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Publication number | Priority date | Publication date | Assignee | Title |
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JP2021097700A (ja) * | 2012-03-26 | 2021-07-01 | アクセラ・ヘルス・インコーポレイテッドAxcella Health Inc. | 荷電栄養タンパク質および方法 |
JP7303238B2 (ja) | 2012-03-26 | 2023-07-04 | アクセラ・ヘルス・インコーポレイテッド | 荷電栄養タンパク質および方法 |
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US20140212541A1 (en) | 2014-07-31 |
JP2021097700A (ja) | 2021-07-01 |
US20240150406A1 (en) | 2024-05-09 |
EP2831098A4 (en) | 2016-12-07 |
CN104470946A (zh) | 2015-03-25 |
HK1206370A1 (en) | 2016-01-08 |
AU2017200234B2 (en) | 2017-06-08 |
SG10201604464SA (en) | 2016-07-28 |
US20140342978A1 (en) | 2014-11-20 |
EP2831098A1 (en) | 2015-02-04 |
US20170246244A1 (en) | 2017-08-31 |
SG11201405842PA (en) | 2014-10-30 |
US20130296231A1 (en) | 2013-11-07 |
JP2018139589A (ja) | 2018-09-13 |
RU2014143029A (ru) | 2016-05-20 |
MX2014011459A (es) | 2015-02-04 |
JP2015519879A (ja) | 2015-07-16 |
CA2868522A1 (en) | 2013-10-03 |
WO2013148329A1 (en) | 2013-10-03 |
IL234621A0 (en) | 2014-11-30 |
US8822412B2 (en) | 2014-09-02 |
AU2013240183A1 (en) | 2014-10-09 |
US8809259B2 (en) | 2014-08-19 |
AU2013240183B2 (en) | 2016-10-20 |
BR112014023869A2 (pt) | 2017-07-18 |
US20200109175A1 (en) | 2020-04-09 |
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