JP2021534763A - 微生物におけるタンパク質のグリコシル化の修飾 - Google Patents
微生物におけるタンパク質のグリコシル化の修飾 Download PDFInfo
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Abstract
Description
本出願は、2018年8月21日出願の米国仮特許出願第62/720,785号(代理人整理番号49160−712.101)の利益を主張する。その開示全体は参照により本明細書に取り込まれる。
参照による組み込み
本明細書中で言及された全ての刊行物、特許および特許出願は、各個々の刊行物、特許または特許出願が具体的かつ個々に参照によって取り込まれると示されるのと同じ程度まで、参照によって本明細書に組み込まれる。
組換えタンパク質を含む組成物
異種発現したアルファ−1,2−マンノシダーゼを保有する微生物
ER標的化/保持シグナル
ベクターによる微生物宿主の形質転換
形質転換された株の有効性の決定
アルファ−1,2−マンノシダーゼの同定
BlastPを使用して、Picha sp.(現在Komagataella種として再分類されている)中で異種発現されたタンパク質上のグリカン構造の修飾を付与することができる公知のアルファ−1,2−マンノシダーゼとの同一性を有するタンパク質配列を検索した。同定された例示的真菌のアルファ−1,2−マンノシダーゼタンパク質配列は、配列番号1〜10を含む。Gallus gallus中の配列についてさらなる検索を実施した。例示的Gallus gallusのアルファ−1,2−マンノシダーゼタンパク質配列は、配列番号145〜150を含む。
(実施例2)
Pichia中のアルファ−1,2−マンノシダーゼの発現のための発現ベクターの構築
(実施例3)
Pichia中のアルファ−1,2−マンノシダーゼの発現
(実施例4)
Pichia中のTrMDS2の異種発現の活性解析
Pichia中のGgMAN1A1の異種発現
(実施例6)
Pichia中のGgMAN1A1の局在化
(実施例7)
HsORM1の脱グリコシル化
最初の誘導実験に続いて、HsORM1+/TrMDS2共発現剤のサブセットを、HsORM1の脱グリコシル化度について比較した(下の図10)。左から右へ、PCRで遺伝子型を決定した株(TrMDS2構築物について陽性)は、SDS−PAGEでより小さなHsORM1ポリペプチド種における増加によって観察されるように、極めて僅かから顕著な脱グリコシル化まで変動するHsOrm1の脱グリコシル化のレベルを呈した。これらの株の比較により、発現した動物タンパク質(HsOrm1等)の脱グリコシル化の程度は、TrMDS2の異なるレベルの発現によって創製されるような種々のレベルの脱グリコシル化パターンを選択することによって微調整できることが示された。
(実施例8)
オボアルブミン(OVA)の脱グリコシル化
(実施例9)
TrMDS1の試験
(実施例10)
OVDのグリコシル化パターンの比較
Claims (41)
- 消費可能な組成物を生産する方法であって、
a.宿主細胞中で栄養タンパク質を組換え発現するステップであって、前記栄養タンパク質が、前記宿主細胞の外に分泌される、ステップ、
b.前記宿主細胞中でα−1,2−マンノシダーゼを組換え発現するステップであって、前記α−1,2−マンノシダーゼが、前記宿主細胞から分泌された前記栄養タンパク質のグリコシル化を、50%を上回って低減し、前記栄養タンパク質が、少なくとももう1つの成分と混合されて前記消費可能な組成物を形成する、ステップ
を含む、方法。 - 前記α−1,2−マンノシダーゼが、配列番号7の配列、その機能的等価物、または配列番号7と85%もしくはそれより高度に同一な配列を有する、請求項1に記載の方法。
- 前記α−1,2−マンノシダーゼが、配列番号150の配列、その機能的等価物、または配列番号150と85%もしくはそれより高度に同一な配列を有する、請求項1に記載の方法。
- 前記消費可能な組成物の栄養含量が、対照組成物の栄養含量と同等またはそれより多く、前記対照組成物が、天然源から単離された同じタンパク質または前記α−1,2−マンノシダーゼによって修飾されていない組換え栄養タンパク質を使用して生産される、請求項1から3に記載の方法。
- 前記栄養含量が、前記組成物のタンパク質含量である、請求項4に記載の方法。
- 前記消費可能な組成物の前記タンパク質含量が、前記対照組成物より少なくとも5%高い、請求項5に記載の方法。
- 前記消費可能な組成物の前記タンパク質含量が、前記対照組成物より少なくとも10%高い、請求項5に記載の方法。
- 前記消費可能な組成物の前記タンパク質含量が、前記対照組成物より少なくとも20%高い、請求項5に記載の方法。
- 前記宿主細胞から分泌された前記栄養タンパク質の少なくとも75%が、修飾されたグリコシル化パターンを有する、請求項1から5に記載の方法。
- 前記宿主細胞から分泌された前記栄養タンパク質の少なくとも80%が、修飾されたグリコシル化パターンを有する、請求項1から5に記載の方法。
- 前記宿主細胞から分泌された前記栄養タンパク質の少なくとも90%が、修飾されたグリコシル化パターンを有する、請求項1から5に記載の方法。
- 前記栄養タンパク質の熱安定性が、対照組成物と比較して増加し、前記対照組成物が、天然源から単離された同じタンパク質または前記α−1,2−マンノシダーゼによって修飾されていない前記組換え栄養タンパク質を使用して生産される、請求項1から11に記載の方法。
- 前記宿主細胞がPichia pastorisである、請求項1から12に記載の方法。
- 前記栄養タンパク質の窒素の炭素に対する比が、その天然源から単離された栄養タンパク質の比と同等またはそれより大きい、請求項1に記載の方法。
- 前記栄養タンパク質が動物タンパク質である、請求項1から14に記載の方法。
- 前記栄養タンパク質が鳥類のタンパク質である、請求項1から14に記載の方法。
- 前記栄養タンパク質が卵白タンパク質である、請求項16に記載の方法。
- 請求項1から17のいずれかに記載の方法を使用して生産される、消費可能な組成物。
- 飲料である、請求項18に記載の消費可能な組成物。
- 食料品である、請求項18に記載の消費可能な組成物。
- 組換え栄養タンパク質の発現のために使用される宿主細胞であって、
a.栄養タンパク質の発現を駆動する第1のプロモーター、
b.配列番号7もしくは150の配列、その機能的等価物、または配列番号7もしくは150と85%もしくはそれより高度に同一な配列を有するα−1,2−マンノシダーゼの発現を駆動する第2のプロモーターを含み、
前記栄養タンパク質のマンノシル化が、前記α−1,2−マンノシダーゼの発現の結果として低減される、宿主細胞。 - 真菌または酵母である、請求項21に記載の宿主細胞。
- Pichia pastorisである、請求項22に記載の宿主細胞。
- 前記栄養タンパク質および前記α−1,2−マンノシダーゼが、1つまたは複数の発現カセットを使用して発現される、請求項21に記載の宿主細胞。
- 前記栄養タンパク質および前記α−1,2−マンノシダーゼが、別個の発現構築物で発現される、請求項24に記載の宿主細胞。
- 前記栄養タンパク質が、前記宿主細胞の外に分泌される、請求項21から25に記載の宿主細胞。
- 前記分泌された栄養タンパク質が、対照組成物と比較して同等のまたはそれより多い栄養含量を有し、前記対照組成物が、天然源から単離された同じタンパク質または前記α−1,2−マンノシダーゼによって修飾されていない組換え栄養タンパク質を使用して生産される、請求項26に記載の宿主細胞。
- 前記栄養含量が、前記タンパク質含量である、請求項27に記載の宿主細胞。
- 前記分泌された栄養タンパク質が、様々な程度のグリコシル化を有する、請求項26から28のいずれか一項に記載の宿主細胞。
- 前記分泌された栄養タンパク質の少なくとも50%が、修飾されたグリコシル化パターンを有する、請求項26から28のいずれか一項に記載の宿主細胞。
- 異種宿主細胞中で産生された組換え動物タンパク質および1つまたは複数の追加の成分を含む消費可能な組成物であって、
前記動物タンパク質が、消費可能な組成物における使用に適したレベルのグリコシル化を含み、前記動物タンパク質が、前記消費可能な組成物に1つまたは複数の食料機能的特徴を提供する、消費可能な組成物。 - 栄養タンパク質をコードする第1の核酸およびα−1,2−マンノシダーゼをコードする第2の核酸を含む微生物であって、
前記α−1,2−マンノシダーゼが、前記微生物に対して異種であり、前記α−1,2−マンノシダーゼが、前記栄養タンパク質のグリコシル化構造を修飾することが可能である、微生物。 - 前記栄養タンパク質が、食料成分として、または食品として使用される、請求項32に記載の微生物。
- 前記α−1,2−マンノシダーゼが、配列番号150、配列番号7、またはそれらに対して80%より高い相同性を有する配列のアミノ酸配列を含む、請求項32に記載の微生物。
- 前記第1および第2の核酸配列が、1つまたは複数の発現カセットに含有される、請求項32に記載の微生物。
- Pichia種である、請求項32に記載の微生物。
- 前記α−1,2−マンノシダーゼがGallus gallusのα−1,2−マンノシダーゼである、請求項32に記載の微生物。
- 前記α−1,2−マンノシダーゼがTrichoderma reeseiのα−1,2−マンノシダーゼであり、前記微生物がPichia種である、請求項32に記載の微生物。
- 前記栄養タンパク質が卵白タンパク質である、請求項32から38のいずれかに記載の微生物。
- 前記卵白タンパク質が、配列番号11〜26のいずれか1つのアミノ酸配列またはそれに対して80%を上回る相同性を有する任意の配列を含む、請求項39に記載の微生物。
- 前記核酸配列の少なくとも1つが前記微生物中における発現のためにコドン最適化されている、請求項32から40のいずれかに記載の微生物。
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MX2017006127A (es) | 2014-11-11 | 2017-11-08 | Clara Foods Co | Metodos y composiciones para produccion de proteina de clara de huevo. |
MX2022000374A (es) | 2019-07-11 | 2022-03-25 | Clara Foods Co | Composiciones de proteina y productos consumibles de las mismas. |
FI13395Y1 (fi) * | 2019-07-11 | 2023-06-06 | Clara Foods Co | Koostumuksia, jotka käsittävät rekombinanttia ovomukoidiproteiinia |
US10927360B1 (en) | 2019-08-07 | 2021-02-23 | Clara Foods Co. | Compositions comprising digestive enzymes |
EP4297581A1 (en) * | 2021-02-23 | 2024-01-03 | Clara Foods Co. | Compositions for preparing animal-free egg-like products |
EP4373289A1 (en) | 2021-07-23 | 2024-05-29 | Clara Foods Co. | Purified protein compositions and methods of production |
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EP1297172B1 (en) * | 2000-06-28 | 2005-11-09 | Glycofi, Inc. | Methods for producing modified glycoproteins |
IL165717A0 (en) * | 2002-06-26 | 2006-01-15 | Flanders Interuniversity Inst | A strain of methylotrophic yeast for producing proteins |
US7507573B2 (en) * | 2003-11-14 | 2009-03-24 | Vib, Vzw | Modification of protein glycosylation in methylotrophic yeast |
CN1746302A (zh) * | 2005-07-18 | 2006-03-15 | 山东大学 | 利用酵母生产非n-糖基化蛋白的方法 |
CN1332025C (zh) * | 2005-07-18 | 2007-08-15 | 山东大学 | 制备基因工程固定化酶n-糖酰胺酶的方法 |
EP1954815B1 (en) * | 2005-11-15 | 2015-02-25 | GlycoFi, Inc. | Production of glycoproteins with reduced o-glycosylation |
ES2434494T3 (es) * | 2005-12-08 | 2013-12-16 | Amgen, Inc. | Células huésped mejoradas y métodos de cultivo |
CN102648286A (zh) * | 2009-10-16 | 2012-08-22 | 默沙东公司 | 在巴斯德毕赤氏酵母中生产缺乏与针对宿主细胞抗原的抗体的可检测交叉结合活性的蛋白质的方法 |
US20120135461A1 (en) * | 2010-07-30 | 2012-05-31 | William James Cook | Production of glycoproteins with reduced o-glycosylation comprising the use of an alpha-1,2-mannosidase |
SG190321A1 (en) * | 2010-11-24 | 2013-06-28 | Novartis Int Pharm Ltd | Fusion enzymes having n-acetylglucosaminyltransferase activity |
CN104936466A (zh) * | 2012-11-20 | 2015-09-23 | 普罗努塔利亚公司 | 工程化的分泌蛋白质和方法 |
WO2016012468A1 (en) * | 2014-07-21 | 2016-01-28 | Novartis Ag | Production of glycoproteins with mammalian-like n-glycans in filamentous fungi |
MX2017006127A (es) * | 2014-11-11 | 2017-11-08 | Clara Foods Co | Metodos y composiciones para produccion de proteina de clara de huevo. |
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CN112888315A (zh) | 2021-06-01 |
JP2024053044A (ja) | 2024-04-12 |
US20210337826A1 (en) | 2021-11-04 |
EP3840582A1 (en) | 2021-06-30 |
WO2020041483A1 (en) | 2020-02-27 |
EP3840582A4 (en) | 2022-08-03 |
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