JP5982504B2 - 一倍体接合方法を用いた酵母におけるヘテロ多量体のポリペプチド類の合成方法 - Google Patents
一倍体接合方法を用いた酵母におけるヘテロ多量体のポリペプチド類の合成方法 Download PDFInfo
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Description
組み換えたんぱく質生産は、高速大量処理スクリーニング、機能性の検証、構造生物学および医薬ポリペプチド類の生産にとっては必須な活動である。Escherichia coli(大腸菌)は、低価格の基質において高細胞密度まで容易に増殖し、確立した遺伝手法および発現ベクター類を有するので、異種性たんぱく質の発現に広く使用される生物である。しかしながら、大腸菌は活性な生物分子を効率的に生産するには必ずしも十分ではない。生物的活性であるためには、ポリペプチド鎖はジスルフィド結合群の適切な形成を含めて正確に生来の三次元構造に折り畳まれていなければならず、複数の鎖の正確な会合を更に必要とする。
形質転換受容性酵母を接合することで組み換えヘテロ多量体たんぱく質群の合成と分泌するための方法が提供される。関心のあるヘテロ多量体たんぱく質群は少なくとも2種の非同一ポリペプチド鎖群、例えば抗体の重鎖および軽鎖群、MHCのアルファおよびベータ鎖群などを含む。発現ベクターは各非同一ポリペプチド鎖用に提供される。
組み換えヘテロ多量体たんぱく質は接合形質転換受容性酵母の二倍体菌株から分泌される。遺伝的に標識した酵母一倍体細胞の対はヘテロ多量体たんぱく質のサブユニットを含む発現ベクター群により形質転換される。1個の一倍体細胞は最初の発現ベクターを含み、別の一倍体細胞は別の発現ベクターを含む。場合によりヘテロ三量体、ヘテロ四量体などの合成のために、更なる発現ベクターを一倍体または二倍体細胞に導入してもよいし;或いは当該最初または別の発現ベクターは更なるコード配列群を含むことができる。非同一ポリペプチドの発現レベルは適当な選択、ベクター複製物数、プロモーターの強さおよび/または誘導などにより個々に較正され、調整される。当該形質転換した一倍体細胞群は遺伝的に交雑させるか融合させる。得られた二倍体または四倍体菌株は完全に組み立てられそして生物的機能性ヘテロ多量体たんぱく質を産生し、分泌するために利用される。
本発明は特定の方法論、手順、細胞菌株、動物種または属および記載した試薬に限られることはなく、変化してもよいと理解すべきものである。また本明細書で使用される専門用語は説明する特定の実施形態のみを説明する目的のためで、本発明の目的を制限する意図は無く、本発明は添付した請求項のみに制限されるであろう。
形質転換した接合形質転換受容性一倍体酵母細胞群は、望ましいたんぱく質のサブユニット対形成を可能とする遺伝的方法を提供する。一倍体酵母菌株群は2つの発現ベクターのそれぞれにて形質転換され、最初のベクターは1つのポリペプチド鎖の合成を導き、別のベクターは別の非同一ポリペプチド鎖の合成を導く。当該2つの一倍体菌株を接合させて、最適化標的たんぱく質産生が得られる二倍体宿主を供する。
抗体遺伝子群:キメラヒト化マウスモノクローナル抗体OKT3の3種類の形態の合成を目指し、遺伝子群をクローン化し、組み立てた。これらの当該組立物において使用する可変領域の出所はGenbankで見出すことができる。受入番号A22261;マウスOKT3重鎖(International Patent Application WO 9109967‐A 3 11‐JUL‐1991)。受入番号A22259;マウスOKT3軽鎖(International Patent Application WO 9109967‐A 3 11‐JUL‐1991)。
軽鎖および重鎖抗体遺伝子群の転写用pGAPZ‐アルファ発現ベクター群の作成。軽鎖および重鎖の両方の可変領域をクローン化するために、マウスOKT3 CD3ハイブリドーマの細胞菌株の細胞群を増殖させ、全RNAを抽出した。その後2種のRT‐PCR反応を実施したが、1種はOKT3抗体遺伝子群の軽鎖に特異的であり、1種はOKT3抗体遺伝子群の重鎖可変領域コード化配列群に特異的であった。当該重鎖および軽鎖可変領域を増幅するために使用したプライマー群はそれぞれの可変領域群に関して(SEQ ID NO:1)5'‐CCGCTCGAGAAAAGAGAGGCTGAAGCTCAGGTCCAGCTGCAGCAGTC‐3'および(SEQ ID NO:3)5'‐CCGCTCGAGAAAAGAGAGGCTGAAGCTCAAATTGTTCTCACCCAGTCTCC‐3'とともに(SEQ ID NO:2)5'‐TGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGTGGTGC‐3'および(SEQ ID NO:4)5'‐GACAGATGGTGCAGCCACAGCCCGG TTTATTTCCAACTTTGTCC‐3'であった。
Claims (9)
- ピキア パストリス(Pichia pastoris)二倍体細胞培養物中に少なくとも2種の非同一サブユニットポリペプチド鎖を含んでいる分泌されたヘテロ多量体たんぱく質の合成回収方法であって、
第一のピキア パストリス一倍体細胞を、第一の酵母プロモーターに作動可能に結合されている、前記たんぱく質の第一のサブユニットを含む第一の発現ベクターにより形質転換させ;
第二のピキア パストリス一倍体細胞を、第二の酵母プロモーターに作動可能に結合されている、前記たんぱく質の第二のサブユニットを含む第二の発現ベクターにより形質転換させ;
前記第一および前記第二のピキア パストリス一倍体細胞から二倍体ピキア パストリス細胞を産生させ;
培養物中ピキア パストリス細胞の濃度が少なくとも50g/Lで、前記第一および前記第二のサブユニットが前記ヘテロ多量体たんぱく質として安定して発現、分泌される条件下で前記二倍体ピキア細胞を培養培地中で培養し;そして
培養培地中から分泌されたヘテロ多量体たんぱく質を回収することを含む、
方法。 - 培養物中ピキア パストリス細胞の濃度が少なくとも100g/Lである、請求項1に記載の方法。
- 培養物中ピキア パストリス細胞の濃度が少なくとも200g/Lである、請求項1に記載の方法。
- 培養物中ピキア パストリス細胞の濃度が少なくとも300g/Lである、請求項1に記載の方法。
- 培養物中ピキア パストリス細胞の濃度が少なくとも400g/Lである、請求項1に記載の方法。
- 培養物中ピキア パストリス細胞の濃度が少なくとも500g/Lである、請求項1に記載の方法。
- 培養物中ピキア パストリス細胞の濃度が500g/Lを超える、請求項1に記載の方法。
- ピキア パストリス培養物中、前記培養物中ヘテロ多量体たんぱく質が、少なくとも500mg/L発現している、請求項1に記載の方法。
- ピキア パストリス培養物中、前記培養物中ヘテロ多量体たんぱく質が、1000mg/Lを超えて発現している、請求項1に記載の方法。
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JP2012029170A Pending JP2012095669A (ja) | 2003-10-22 | 2012-02-14 | 一倍体接合方法を用いた酵母におけるヘテロ多量体のポリペプチド類の合成方法 |
JP2015000021A Active JP5982504B2 (ja) | 2003-10-22 | 2015-01-05 | 一倍体接合方法を用いた酵母におけるヘテロ多量体のポリペプチド類の合成方法 |
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JP2012029170A Pending JP2012095669A (ja) | 2003-10-22 | 2012-02-14 | 一倍体接合方法を用いた酵母におけるヘテロ多量体のポリペプチド類の合成方法 |
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US (7) | US8268582B2 (ja) |
EP (2) | EP2330201B1 (ja) |
JP (3) | JP4994038B2 (ja) |
CN (2) | CN101979650B (ja) |
AU (2) | AU2004283299B2 (ja) |
CA (1) | CA2541651C (ja) |
DK (2) | DK1678314T3 (ja) |
ES (2) | ES2393555T3 (ja) |
HK (1) | HK1093222A1 (ja) |
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