JP4969068B2 - タンパク質の高発現システム - Google Patents
タンパク質の高発現システム Download PDFInfo
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- JP4969068B2 JP4969068B2 JP2005211454A JP2005211454A JP4969068B2 JP 4969068 B2 JP4969068 B2 JP 4969068B2 JP 2005211454 A JP2005211454 A JP 2005211454A JP 2005211454 A JP2005211454 A JP 2005211454A JP 4969068 B2 JP4969068 B2 JP 4969068B2
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- 108090000623 proteins and genes Proteins 0.000 title claims description 19
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- 230000000694 effects Effects 0.000 description 31
- 101710191666 Lactadherin Proteins 0.000 description 20
- 102100039648 Lactadherin Human genes 0.000 description 19
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- 108010005774 beta-Galactosidase Proteins 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
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- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 101100327917 Caenorhabditis elegans chup-1 gene Proteins 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
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- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
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Description
まず本発明者らはDNAマイクロアレイを用いて、清酒醪における酵母の全遺伝子について経時的な発現量を定量し、網羅的なプロモーター検索を実施した。ここでヒットしたプロモーターについて詳細な転写活性を調べる方法としては、対象プロモーターの下流域にレポーター遺伝子を連結し、そのレポーター遺伝子産物の酵素活性をプロモーター発現の指標とする方法を採用した。そして各種遺伝子のプロモーターについて検討した結果、好適なプロモーターを発見し、さらに研究の結果、遺伝子高発現システムの創製に成功した。
本発明に係る高発現プロモーターを検索するために、Research Genetics社の酵母用DNAマイクロアレイ「GeneFilters」を用いた。具体的には以下の操作を行なった。一般的な配合で仕込んだ清酒醪から経時的に酵母total RNAを抽出した。このRNAを鋳型に放射ラベル化したcDNAプローブを作製したのち、GeneFiltersとハイブリダイズさせた。各遺伝子のスポット強度を定量し、データポイント約30万点にのぼる酵母遺伝子発現データベースを構築した。
まず、プロモーター解析を行うについては、SED1プロモーターの下流域にレポーター遺伝子を連結し、そのレポーター遺伝子産物の活性をプロモーター発現の指標とする方法を採用した。
その結果、SED1プロモーターを−800塩基まで欠失させた場合、元の−1063塩基までのSED1プロモーター活性に対して、9時間培養では活性に変化がないが、24時間培養の場合では、2倍以上のβ−ガラクトシダーゼ活性が得られた。従って、−1063塩基から−800塩基の間に後期抑制因子があると考えられた。
特願2002−69198(特開2003−265177)において、本発明者らはSED1プロモーターが強力なプロモーター活性を持つことを明らかにした。本発明において、1063塩基のプロモーターのうち、シスエレメントを究明すべく、デリーション解析を行い、より強力なプロモーターを探索することにした。
元のプロモーターの長さが−1063塩基までであるSED1プロモーターを、それぞれ−800塩基まで、−600塩基まで、−400塩基まで、−200塩基まで欠失したプロモーターを作成し、前述したプラスミドを構築した。構築したプラスミドを清酒酵母協会7号の栄養要求性株に形質転換し、導入プラスミドが宿主染色体のura3位に1コピー組み込まれた形質転換体を選択した。そしてこれら形質転換体の細胞破砕上清のβ−ガラクトシダーゼ(LacZ)活性を測定することにより、プロモーター活性の指標とした。
酵母培養時間9時間、24時間の時点でそれぞれβ−ガラクトシダーゼ(LacZ)活性を測定した。その結果を表1に示す。
元の1063塩基のSED1プロモーター、及び実施例1で得た800塩基までデリーションしたSED1プロモーターを用いて、実施例1と同じ方法で形質転換体に導入し、ジャーファーメンター培養で、48時間までの培養を行った。そのβ−ガラクトシダーゼ(LacZ)活性を測定した。その結果を図2に示す。
Claims (1)
- 配列表の配列番号1に示す塩基配列からなるプロモーターDNAと有用タンパク質DNAからなる融合遺伝子DNAを含むプラスミドDNAを、酵母に移入してなる形質転換体を、培地で24時間以上培養し、当該培養物から有用タンパク質を得ることを特徴とする有用タンパク質の生産方法。
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JP2007020538A JP2007020538A (ja) | 2007-02-01 |
JP2007020538A5 JP2007020538A5 (ja) | 2010-04-08 |
JP4969068B2 true JP4969068B2 (ja) | 2012-07-04 |
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ES2616428T3 (es) | 2007-12-07 | 2017-06-13 | Toray Industries, Inc. | Casete de expresión para lactasa deshidrogenasa, levadura transformada y método para producir ácido láctico |
JP6478375B2 (ja) * | 2014-08-01 | 2019-03-06 | 国立研究開発法人産業技術総合研究所 | 高発現プロモーター遺伝子 |
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JP2003265177A (ja) * | 2002-03-13 | 2003-09-24 | Gekkeikan Sake Co Ltd | タンパク質の高発現システム |
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