JP3905695B2 - Fission yeast glycosyltransferase gene och1 - Google Patents
Fission yeast glycosyltransferase gene och1 Download PDFInfo
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- JP3905695B2 JP3905695B2 JP2000263266A JP2000263266A JP3905695B2 JP 3905695 B2 JP3905695 B2 JP 3905695B2 JP 2000263266 A JP2000263266 A JP 2000263266A JP 2000263266 A JP2000263266 A JP 2000263266A JP 3905695 B2 JP3905695 B2 JP 3905695B2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
【0001】
【発明の属する技術分野】
本発明は、分裂酵母の糖転移酵素をコードするDNA、及び該DNAの機能を欠損させることにより蛋白質への糖鎖付加を行なう能力が減少させられた分裂酵母の製造、該分裂酵母を利用した糖鎖付加の少ない外来蛋白質を産生させる方法に関する。
【0002】
【従来の技術】
酵母は動物細胞と比較して、分裂速度が速く、安価な培地で生育が可能なこと、また細菌と比較して、翻訳後修飾が動物細胞と類似していること、特に細菌では付加しない糖鎖が付加することから、組換えDNA技術で得られたDNAを用いた蛋白質産生の宿主として注目を集めた。
しかし、動物細胞と酵母では一部の糖転移酵素が異なり、従って酵母で産生された蛋白質に、動物細胞で見られない大量のマンノースから成る糖外鎖(以下高マンノース型糖鎖と称す)が付加する場合が多く経験された。そして高マンノース型糖鎖の付加は、蛋白質の生産性、蛋白質の抗原性や体内動態等に影響を及ぼし、酵母での組換え蛋白質生産の大きな問題点となっていた。
【0003】
酵母は出芽酵母と分裂酵母に大きく分類され、出芽酵母においては、高マンノース型糖鎖を付加する糖転移酵素遺伝子OCH1が見出され(Nagasu, T., et.al., Yeast, 8, 535-547, 1992)、OCH1を欠失した酵母株を用いて組換え蛋白質を産生させると高マンノース型の糖鎖が付加しないことが見出された。しかし、分裂酵母においてはハイブリダイゼーション等の通常の方法では出芽酵母のOCH1に対するホモログが見つからず、高マンノース型の糖鎖が付加しない変異体を作製することはできなかった。
【0004】
【発明が解決しようとする課題】
本発明の課題は、分裂酵母の糖転移酵素をコードする遺伝子を特定し、その遺伝子の機能を失わせた分裂酵母を使って糖鎖付加の少ない蛋白質を産生させることにある。
【0005】
【課題を解決するための手段】
本発明者らは、高温においては浸透圧を調整した条件でしか生育できない分裂酵母変異体に、分裂酵母のゲノムDNAライブラリーを導入し、その性質を相補できるゲノムDNA断片から、出芽酵母のOCH1とホモロジーがある配列番号1に記載の塩基配列からなる遺伝子を見出した。その遺伝子は分裂酵母の糖転移酵素をコードする遺伝子であると考えられ、分裂酵母からその遺伝子を欠失させたところ、欠失させない分裂酵母では付加していた高マンノース型の糖鎖が付加しなくなったことを見出し、本発明を完成するに到った。
【0006】
すなわち本発明は、下記の(a)から(c)のいずれかに記載のDNA(以下該DNAを「och1+」と記載する)。
(a)配列番号2に記載のアミノ酸配列からなる蛋白質をコードするDNA。
(b)配列番号1に記載の塩基配列からなるDNA。
(c)配列番号1に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、糖転移酵素活性を有する蛋白質をコードする分裂酵母由来のDNA、およびoch1+がコードする蛋白質(以下「Och1」蛋白と称す)に関する。
【0007】
ここでストリンジェントな条件の一例は、65℃ 4 x SSCにおけるハイブリダイゼーション、次いで65℃で1時間0.1 x SSC中での洗浄である。また別法としてストリンジェントな条件は、50%ホルムアミド中42℃ 4 x SSCである。
【0008】
また本発明は、och1+あるいはその一部を用いて、分裂酵母遺伝子中のoch1+の機能を失わせる方法に関する。
【0009】
ここで、「och1+の機能を欠損させる」には、och1+の蛋白質をコードする領域を変異させて、Och1蛋白の機能を失わせること、およびoch1+の発現を減少させることの双方が含まれる。
「Och1蛋白の機能を失わせる」とは、分裂酵母が産生する蛋白質の大部分に過剰な高マンノース型の糖鎖が付加しないようにOch1蛋白が改変されていることを意味する。
また、「och1+の発現を減少させる」とは、分裂酵母のゲノムにおけるoch1+またはその発現制御領域に変異を導入することにより、該分裂酵母が産生するOch1蛋白量を、該変異を導入しない分裂酵母が産生するOch1蛋白量と比較して、少なくさせることを指す。
【0010】
更に本発明は、och1+の機能を欠損させることにより糖鎖付加が減少された蛋白質を産生する能力を有する分裂酵母(以下och1+破壊株と称す)およびその製造方法、該方法により製造し得る分裂酵母、好ましくはSchizosaccharomyces pombe、該分裂酵母(och1+破壊株)に、糖鎖付加の少ない蛋白質を産生させる方法、及びその方法により産生された糖鎖付加の少ない蛋白質に関する。
【0011】
【発明の実施の形態】
以下に本発明の実施の形態について詳細に説明する。
【0012】
och1+あるいはその一部を含むDNAは、配列番号1に記載の塩基配列を基にプライマーを設定し、分裂酵母ゲノムDNAあるいはcDNAを鋳型にPCRを行なうことにより、あるいは分裂酵母RNAを鋳型にRT-PCRを行なうことにより得ることができる。また別法としては、配列番号1に記載の塩基配列を基にプローブを合成して、分裂酵母のゲノムDNAライブラリーあるいはcDNAライブラリーより、プローブとハイブリダイズするクローンを選び出し、塩基配列を決定して、och1+あるいはその一部を含むクローンを選択しても良い。
【0013】
更に、Och1蛋白の発現に影響を与えるoch1+の発現制御領域は、配列番号1に記載の塩基配列を基にプローブを合成して、分裂酵母ゲノムDNAライブラリーより、プローブとハイブリダイズするクローンを選び出し、塩基配列を決定して、och1+の上流あるいは下流配列を含むクローンを選択することにより得ることができる。判明した上流あるいは下流の塩基配列を基にプローブを合成して、更に上流あるいは下流配列を含むクローンを選択しても良い。
【0014】
発現制御活性を有する領域は、通常コード領域の上流1 kbp、下流500 bpの中に存在する。相同組換えにより単離した領域を欠失させた分裂酵母においてノーザンブロッティングにより定量されるoch1+ mRNA量が減少しているか、あるいは該分裂酵母の産生する蛋白質に過剰な高マンノース型の糖鎖付加が無くなっているかを解析することにより、単離した領域が発現制御活性を有するか否かを判定することができる。
【0015】
得られたoch1+遺伝子は、発現ベクターに挿入してOct1蛋白質を産生させることができる。発現ベクターは、宿主が大腸菌であれば、例えばpGEX、宿主が酵母であれば、例えばpcL, pREP、宿主が哺乳類細胞であれば、例えばpCMV, pSRα等に挿入する。挿入は例えばSambruck, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYに記載の方法に従って行うことができる。Oct1蛋白質は、His6, GSTとの融合蛋白として発現させると、後の精製が容易になる点で望ましい。
【0016】
och1+遺伝子を挿入した発現ベクターは、宿主に適した方法で宿主に導入する。導入は、例えば、Sambruck, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYに記載の方法に従って行うことができる。
【0017】
och1+遺伝子を導入した宿主を適当な条件で培養した後、宿主を破壊し、蛋白質画分を抽出する。破砕は、宿主が大腸菌や酵母であればガラスビーズあるいは超音波を用いて、宿主が哺乳類細胞であればホモゲナーザーを用いて行なうことができる。発現ベクターに産生される蛋白質が培養上清に分泌されるよう工夫が施されている場合は、培養上清から目的の蛋白質を回収することができる。
【0018】
抽出した蛋白質画分より、電気泳動、種々のクロマトグラフィー、例えばアフィニティーカラム、イオン交換カラム、ゲルフィルトレーションカラム等を用いた標準的な方法によりoch1+遺伝子産物を精製できる。導入した遺伝子が、融合蛋白質をコードするように設計されている場合、例えばGSTの場合はグルタチオンカラムにより、His6の場合はニッケルセファロースカラムを用いて容易に精製できる。
【0019】
och1+の蛋白質をコードする領域を変異させる方法は、分裂酵母遺伝子中のoch1+と、変異を導入したoch1+あるいはその一部を含む環状又は線状DNAとの間で相同組換えを起こさせ、分裂酵母遺伝子中のoch1+を、変異を導入したoch1+に置き換えることを基本とする。組換えに用いられるoch1+の一部を含むDNAは、通常、500 bp以上であり、1 kbp以上であることが望ましい。
【0020】
PCRまたはクローニングにより得られたoch1+あるいはその一部を含むDNAに変異を導入する方法としては、DNAの塩基配列を操作する組換えDNA技術( Sambruck, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY )、PCRを応用した技術(Ling MM. and Robinson BH., Anal. Biochem. 254(2):157-78, 1997)等、多くの方法が適用可能である。例えば適当な制限酵素で切断した後に、無関係な遺伝子、好ましくは相同組換えを起こした分裂酵母を選択できる選択マーカ遺伝子、更に好ましくは選択マーカとしてウラシル合成系の遺伝子ura4+を挿入する方法を挙げることができるが、適当な変異を導入できる方法であれば、この方法に限られるものではない。適当な制限酵素部位が無い場合にはPCR等により、適当な制限酵素部位を入れてもよい。
【0021】
変異を導入したoch1+あるいはその一部を含む、環状又は線状DNAは、スフェロプラスト法、酢酸リチウム法、エレクトロポレーション法等の方法により分裂酵母に導入される。導入した変異遺伝子に選択マーカ、例えばura4+が含まれていれば、選択マーカを持つ分裂酵母のみが生き残る培養条件、例えば選択マーカがura4+であればウラシルを含まない培地中で培養することにより、相同組換えを起こし、変異を導入したoch1+を持った分裂酵母、すなわちoch1+破壊株を選択することができる。
【0022】
Och1蛋白の発現を減少させる方法は、前述のoch1+の蛋白質をコードする領域を含むDNAに代えて、クローニングしたoch1+の発現制御領域(上流あるいは下流配列を含む)を用い、Och1蛋白の発現に影響を与える塩基配列に変異を導入することにより、前述のoch1+の蛋白質をコードする領域の場合と同様に行うことができる。
【0023】
また、Och1蛋白の発現を減少させる方法としては、分裂酵母にoch1+の転写産物に相補的なRNA(アンチセンスRNA)をコードするDNAが挿入された発現ベクターを導入して、Och1蛋白の発現を減少させる等の方法も考えられる。
ここで「相補的」とは、och1+の発現を抑制し得る限り、完全に相補的である場合に限られない。通常、80%以上、好ましくは90%以上、さらに好ましくは95%以上の相補性があれば良い。
【0024】
アンチセンスRNAをコードするDNAは、通常、翻訳開始部位等mRNAの機能に重要な役割を果たす部位と相補的な20 bpの鎖長があれば足りる。しかしながら、高次構造をとることによりアンチセンスとして機能しなくなることを考慮すれば、さらに長い鎖長、好ましくは100 bpの鎖長を用いることも可能である。
【0025】
Och1蛋白の発現を減少させる処理が施され、その結果、産生する蛋白質の大部分に過剰な高マンノース型の糖鎖が付加しなくなっている分裂酵母である限り、本発明の分裂酵母に含まれる。
【0026】
過剰な高マンノース型の糖鎖が付加しているかどうかは、高マンノース型の糖鎖が多様性を示すことから、例えば、高マンノース型の糖鎖が付加した蛋白質が多様な分子量を示すことより推定できる。すなわち、野生型och1+を持った分裂酵母では、電気泳動でスメアなパターンを示す蛋白質が、変異を導入した分裂酵母でシャープなパターンを示せば、過剰な高マンノース型の糖鎖が付加の無い蛋白質が産生されたことを推定することができる。
【0027】
以下に、och1+の機能を失わせる方法の具体的な例を記載する。機能を失わせたoch1+を含む線状DNAとして、例えばoch1+の翻訳領域の中央部約60%をウラシル合成系の遺伝子ura4+に置換した線状DNA(och1::ura4+)を作製する。これをura4+を欠損する分裂酵母に導入して、och1::ura4+と分裂酵母中のoch1+の間で相同組換えを起こさせる。その後ウラシルを含まない培地中で培養して、ura4+を持った、すなわち相同組み換えによりoch1+がoch1::ura4+に置き換わった分裂酵母を選択することにより、och1+の機能を失わせた分裂酵母、すなわちoch1+破壊株を得ることができる。
【0028】
更に本発明は、och1+破壊株を用いて糖鎖付加の少ない蛋白質を産生する方法にも関する。ここで蛋白質は、分裂酵母が産生する蛋白質ならいかなる蛋白質でもよく、分裂酵母由来の遺伝子、分裂酵母由来でない外来遺伝子の何れの遺伝子がコードする蛋白質であってもよい。ここで外来遺伝子とは、分裂酵母で発現させたい遺伝子であり、動物・酵母・細菌等いかなる生物より由来した遺伝子でも構わないが、好ましくは動物由来の遺伝子が望ましい。外来遺伝子は、融合蛋白質等の人工の配列を持っていてもよい。
【0029】
遺伝子の導入法としては、スフェロプラスト法、酢酸リチウム法、エレクトロポレーション法が挙げられるが、本発明はこれに限られるものではない。具体的に説明すると、例えば酢酸リチウム法は、対数増殖期の分裂酵母を酢酸リチウム中で、30℃、1時間保温し、その後導入DNAおよびポリエチレングリコール溶液を加え、30℃、1時間保温する。これを43℃、15分熱処理した後遠心により細胞を回収し培地中に懸濁して、30℃、2時間振とう培養する。この培養液を選択寒天培地上にひろげ、30℃で3日間培養すると、遺伝子が導入され選択培地上で増殖可能な酵母細胞のみがコロニーを形成する。
【0030】
本発明には、och1+破壊株により産生された糖鎖付加の少ない蛋白質も含まれるものである。ここで、本発明に記載の分裂酵母より産生された蛋白質は、培養上清あるいは菌体より、アフィニティーカラム・イオン交換カラム・ゲルフィルトレーションカラム等の標準的な方法により精製できる。導入した遺伝子が、融合蛋白質をコードするように設計されている場合、例えばGSTとの融合蛋白質の場合は、融合蛋白質とのアフィニティーカラム、GSTの場合はグルタチオンカラムにより精製することが好ましい。
【0031】
尚、以上に記載した操作は適当なマニュアル、例えばSambruck, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 等に記載の方法に従って行うことができる。
【0032】
【実施例】
以下に、本発明を下記実施例により更に詳しく説明するが、本発明はこれに限られるものではない。
【0033】
[実施例1] 配列番号1に記載の塩基配列からなる分裂酵母och1+と出芽酵母OCH1のホモロジー
分裂酵母のゲノムDNAライブラリーよりクローニングされた、分裂酵母変異体の高温においては浸透圧を調製した条件でしか生育できない性質を相補できるゲノムDNA断片から、配列番号1に記載の塩基配列からなる分裂酵母の遺伝子och1+を見出した。そして、図1に示すように分裂酵母och1+(SpOCH1)と、出芽酵母OCH1(ScOCH1)との間にホモロジーが見出された。
【0034】
[実施例2] och1+破壊株の作製
変異を導入した配列番号1に記載の塩基配列を含むDNAと、分裂酵母och1+の間で相同組換えを起こさせ、och1+破壊株を作製した。
配列番号1に記載の塩基配列を含むDNAの、och1+の翻訳領域内に存在する2つのHindIII切断部位に挟まれた領域を、分裂酵母ura4+に置換したDNAを作製した(図2)。その後、スフェロプラスト法にて分裂酵母を形質転換し、ウラシルを含まない選択培地で培養して、ura4+を持った、すなわち変異が導入されたoch1+を持った分裂酵母を選択し、och1+破壊株を作製した。
【0035】
[実施例3] och1+を欠失させた分裂酵母の形態
顕微鏡にて、och1+を欠失させた分裂酵母と野生型分裂酵母を比較した。
図3に示すように、och1+を欠失させた分裂酵母は、野生型分裂酵母に比べて丸く短い形態をしている。また、凝集しやすく、増殖速度も遅い。37℃では生育できないという温度感受性を示すが、培地中に1.5%程度のソルビトールを加え高浸透圧条件とすると、37℃でも生育できるようになる。
【0036】
[実施例4] och1+破壊株の培養と、産生された蛋白質の解析
och1+の破壊が、産生される蛋白質の糖鎖付加に対して、影響を与えるかどうかを検討した。
och1+破壊株及び野生株の分裂酵母を培養し、産生される糖タンパク質、ここでは酸性フォスファターゼを電気泳動し、活性染色を行って酸性フォスファターゼの分子量分布を調べた。
その結果、4に示すように、酸性フォスファターゼは野生型株では分子量が大きくスメアーなパターンを示したのに対し(レーン1)、och1+破壊株では分子量が小さくシャープなバンドとなった(レーン2)。このことは多様性に富み分子量の大きなhigh-mannose typeの糖鎖付加が、野生型株では起こり、och1+破壊株では起こっていないことを示唆している。
【0037】
更にこのような電気泳動上の移動度の違いが糖鎖付加によるものかどうかを調べるために、エンドグリコシダーゼH(Endo H)による糖鎖切断処理を行った。エンドグリコシダーゼHはN-結合型糖鎖の根元にある、2つのN-アセチルグルコサミン残基の間を切る酵素であるため、エンドグリコシダーゼH処理された糖蛋白質は、ポリペプチド部分から推定される分子量とほぼ同じ移動度を示す。
この結果、エンドグリコシダーゼH処理したレーン3及び4の野生型由来およびoch1+破壊株由来の酸性フォスファターゼのバンドはどちらも同じところに現れ、レーン1と2の移動度の違いは、N-結合型糖鎖の大きさの違いに起因するものであることが示された。
【0038】
[実施例5] och1+遺伝子産物のマンノース転移酵素活性
och1+遺伝子産物のマンノース転移酵素活性を、蛍光標識したMan9GlcNAc2-PA及びGDP-マンノースを基質とし、och1+破壊株、och1+発現ベクターを導入したoch1+破壊株、及び野生株の菌体を破砕した粗抽出物を酵素として測定した。
分裂酵母och1+遺伝子をpREP1(Maundrell, K. 1993. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123, 127-130)発現ベクターに組み込み、och1+発現ベクターを作製した。och1+発現ベクターを導入しoch1+遺伝子産物を過剰発現させたoch1+破壊株、コントロールとしてベクターのみを導入したoch1+破壊株、及び野生株の菌体を破砕し、100,000 x gで沈殿させたものを酵素として使用した。50 mM Tris HCl (pH 7.5), 10 mM MnCl2, 0.6% Triton X-100, 0.5 mM 1-deoxymannojirimycin, 2 mM PA-sugar chain, 1 mM GDP-マンノースを反応溶液とし、酵素を加えて酵素反応を開始した。30℃で10, 20, 30分間反応後、99℃5分間処理して反応を停止し、反応産物をTSK Gel Amide-80カラムを用いたHPLCにより分離して、酵素反応を解析した。
【0039】
図5に示す通り、och1+破壊株ではMan9GlcNAc2-PAへのマンノースの転移は観察されなかったが、野生株ではマンノースの転移が見られ、och1+発現ベクターを導入しoch1+遺伝子産物を過剰発現させたoch1+破壊株では更に多くのマンノースの転移が見られた。
この結果より、och1+遺伝子産物はマンノース転移酵素活性としての活性を有し、S. cerevisiae OCH1遺伝子産物と同様な反応に関与することが確認された。
【0040】
【発明の効果】
本発明により、糖転移酵素の機能を失わせた分裂酵母の入手が可能となり、分裂酵母においても付加糖鎖の少ない蛋白質を産生することが可能となった。
【0041】
【配列表】
【図面の簡単な説明】
【図1】 分裂酵母och1+と出芽酵母OCH1のホモロジーを示す図である。
【図2】 och1+の中央にura4+を挿入したDNAの構築を示す図である。
【図3】 och1+を欠失させた分裂酵母の形態を示す写真である。
【図4】 och1+破壊株で産生された酸性フォスファターゼの解析結果を示す写真である。
【図5】 och1+遺伝子産物のマンノース転移酵素活性を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a DNA encoding a glycosyltransferase of fission yeast, production of fission yeast in which the ability to add a sugar chain to a protein is reduced by deleting the function of the DNA, and the use of the fission yeast The present invention relates to a method for producing a foreign protein with little glycosylation.
[0002]
[Prior art]
Yeast has a faster division rate than animal cells and can grow on inexpensive media, and has post-translational modifications similar to animal cells compared to bacteria, especially sugars that are not added by bacteria. Due to the addition of strands, it attracted attention as a host for protein production using DNA obtained by recombinant DNA technology.
However, some glycosyltransferases differ between animal cells and yeast, so the protein produced in yeast has an extra-sugar chain consisting of a large amount of mannose that is not found in animal cells (hereinafter referred to as high mannose-type sugar chains). Many cases were added. Addition of a high mannose sugar chain has affected protein productivity, protein antigenicity and pharmacokinetics, and has been a major problem in recombinant protein production in yeast.
[0003]
Yeasts are broadly classified into budding yeast and fission yeast. In budding yeast, a glycosyltransferase gene OCH1 that adds a high mannose-type sugar chain is found (Nagasu, T., et.al., Yeast, 8, 535). -547, 1992), it was found that when a recombinant protein was produced using a yeast strain lacking OCH1, a high-mannose sugar chain was not added. However, in fission yeast, homologues of Saccharomyces cerevisiae to OCH1 were not found by ordinary methods such as hybridization, and it was not possible to produce a mutant to which a high-mannose sugar chain was not added.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to identify a gene encoding a glycosyltransferase of fission yeast and to produce a protein with little glycosylation using the fission yeast in which the function of the gene is lost.
[0005]
[Means for Solving the Problems]
The present inventors introduced a genomic DNA library of fission yeast into a fission yeast mutant that can only grow under conditions where the osmotic pressure is adjusted at high temperatures, and from the genomic DNA fragment that can complement its properties, OCH1 of budding yeast And a gene consisting of the base sequence described in SEQ ID NO: 1 with homology. The gene is thought to be a gene encoding fission yeast glycosyltransferase. When the gene was deleted from fission yeast, a high mannose-type sugar chain added to fission yeast that was not deleted was added. The inventors have found that they have disappeared and have completed the present invention.
[0006]
That is, according to the present invention, the DNA described in any one of (a) to (c) below (hereinafter referred to as “och1 + ”).
(a) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(b) DNA comprising the base sequence set forth in SEQ ID NO: 1.
(c) DNA derived from fission yeast that hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having glycosyltransferase activity, and a protein encoded by och1 + (Referred to as “Och1” protein).
[0007]
An example of stringent conditions here is hybridization at 65 ° C. in 4 × SSC, followed by washing at 65 ° C. for 1 hour in 0.1 × SSC. Alternatively, stringent conditions are 42 ° C. 4 × SSC in 50% formamide.
[0008]
The present invention also relates to a method for losing the function of och1 + in a fission yeast gene using och1 + or a part thereof.
[0009]
Here, “deleting the function of och1 + ” includes both mutating the region encoding the protein of och1 + to lose the function of Och1 protein and decreasing the expression of och1 + It is.
“Loss of the function of Och1 protein” means that the Och1 protein is modified so that excessive high-mannose sugar chains are not added to the majority of the protein produced by fission yeast.
In addition, “decrease the expression of och1 + ” means that the amount of Och1 protein produced by the fission yeast is not introduced by introducing a mutation into the och1 + or its expression control region in the fission yeast genome. This refers to reducing the amount of Och1 protein produced by fission yeast.
[0010]
The present invention further fission yeast (hereinafter referred to as och1 + disrupted strain) and a method of manufacturing the same having the ability glycosylation to produce a reduced protein by lacking the function of och1 +, may be prepared by the method The present invention relates to a fission yeast, preferably Schizosaccharomyces pombe, a method for causing the fission yeast (och1 + disrupted strain) to produce a protein with little glycosylation, and a protein with little glycosylation produced by the method.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
[0012]
For DNA containing och1 + or a part thereof, a primer is set based on the nucleotide sequence shown in SEQ ID NO: 1, and PCR is performed using fission yeast genomic DNA or cDNA as a template, or RT using fission yeast RNA as a template. -It can be obtained by performing PCR. As another method, a probe is synthesized based on the nucleotide sequence shown in SEQ ID NO: 1, a clone that hybridizes with the probe is selected from a genomic DNA library or cDNA library of fission yeast, and the nucleotide sequence is determined. A clone containing och1 + or a part thereof may be selected.
[0013]
Furthermore, the och1 + expression control region that affects the expression of the Och1 protein synthesizes a probe based on the nucleotide sequence shown in SEQ ID NO: 1, and then clones that hybridize with the probe from the fission yeast genomic DNA library. It can be obtained by selecting, determining the base sequence, and selecting a clone containing an upstream or downstream sequence of och1 + . A probe may be synthesized based on the identified upstream or downstream base sequence, and a clone further containing an upstream or downstream sequence may be selected.
[0014]
The region having expression control activity is usually present in 1 kbp upstream and 500 bp downstream of the coding region. In fission yeast lacking a region isolated by homologous recombination, the amount of och1 + mRNA quantified by Northern blotting is reduced, or excessive high-mannose glycosylation is added to the protein produced by the fission yeast It is possible to determine whether or not the isolated region has expression control activity by analyzing whether or not there is a loss.
[0015]
The obtained och1 + gene can be inserted into an expression vector to produce Oct1 protein. The expression vector is inserted into, for example, pGEX if the host is Escherichia coli, for example, pcL or pREP if the host is yeast, or to pCMV, pSRα or the like if the host is a mammalian cell. Insertion can be performed, for example, according to the method described in Sambruck, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Oct1 protein is desirable in that it can be easily purified later when expressed as a fusion protein with His6 and GST.
[0016]
The expression vector into which the och1 + gene has been inserted is introduced into the host by a method suitable for the host. The introduction can be performed, for example, according to the method described in Sambruck, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
[0017]
After culturing the host into which the och1 + gene has been introduced under appropriate conditions, the host is destroyed and the protein fraction is extracted. The disruption can be performed using glass beads or ultrasonic waves if the host is Escherichia coli or yeast, and using a homogenizer if the host is a mammalian cell. In the case where a contrivance is made so that the protein produced in the expression vector is secreted into the culture supernatant, the target protein can be recovered from the culture supernatant.
[0018]
From the extracted protein fraction, the och1 + gene product can be purified by standard methods using electrophoresis, various chromatographies such as affinity columns, ion exchange columns, gel filtration columns and the like. When the introduced gene is designed to encode a fusion protein, it can be easily purified using, for example, a glutathione column in the case of GST and a nickel sepharose column in the case of His6.
[0019]
The method of mutating the och1 + protein coding region is to cause homologous recombination between och1 + in the fission yeast gene and circular or linear DNA containing mutated och1 + or a part thereof. Basically, och1 + in the fission yeast gene is replaced with och1 + introduced with a mutation. The DNA containing a part of och1 + used for recombination is usually 500 bp or more, and preferably 1 kbp or more.
[0020]
As a method for introducing mutation into DNA containing och1 + or a part thereof obtained by PCR or cloning, recombinant DNA technology (Sambruck, J., Fritsch, EF, and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), PCR applied technology (Ling MM. And Robinson BH., Anal. Biochem. 254 (2): 157-78, 1997) etc. are applicable. For example, after cutting with an appropriate restriction enzyme, a selection marker gene capable of selecting an irrelevant gene, preferably a fission yeast that has undergone homologous recombination, and more preferably, a method for inserting the uracil synthesis system gene ura4 + as a selection marker. However, the method is not limited to this method as long as an appropriate mutation can be introduced. If there is no appropriate restriction enzyme site, an appropriate restriction enzyme site may be inserted by PCR or the like.
[0021]
Circular or linear DNA containing och1 + or a part thereof introduced with a mutation is introduced into fission yeast by a method such as a spheroplast method, a lithium acetate method, or an electroporation method. If the introduced mutant gene contains a selection marker, such as ura4 + , culture conditions where only the fission yeast with the selection marker survives, for example, if the selection marker is ura4 + , are cultured in a medium that does not contain uracil. It is possible to select a fission yeast having och1 + that has undergone homologous recombination and introduced a mutation, ie, an och1 + disruption strain.
[0022]
The method of decreasing the expression of Och1 protein uses the cloned och1 + expression control region (including upstream or downstream sequence) instead of the DNA containing the region encoding och1 + protein described above. By introducing a mutation into the base sequence that affects the above, it can be carried out in the same manner as in the region encoding the och1 + protein described above.
[0023]
In addition, as a method of reducing the expression of Och1 protein, expression of Och1 protein is introduced by introducing an expression vector in which DNA encoding RNA (antisense RNA) complementary to och1 + transcript is inserted into fission yeast. It is also possible to consider a method of reducing the value.
Here, “complementary” is not limited to the case of being completely complementary as long as the expression of och1 + can be suppressed. Usually, complementarity of 80% or more, preferably 90% or more, more preferably 95% or more is sufficient.
[0024]
The DNA encoding the antisense RNA usually only needs to have a 20 bp chain length complementary to a site that plays an important role in the function of the mRNA, such as the translation initiation site. However, in view of the fact that it does not function as an antisense due to its higher-order structure, it is possible to use a longer chain length, preferably a 100 bp chain length.
[0025]
It is included in the fission yeast of the present invention as long as it is a fission yeast that has been treated to reduce the expression of Och1 protein, and as a result, excess high-mannose sugar chains are not added to the majority of the protein produced. .
[0026]
Whether excessive mannose-type sugar chains are added or not is determined by the fact that high-mannose-type sugar chains show diversity, for example, proteins added with high-mannose-type sugar chains show various molecular weights. Can be estimated. In other words, in fission yeast with wild-type och1 + , if a protein showing a smear pattern on electrophoresis shows a sharp pattern in a fission yeast into which mutations have been introduced, excess high-mannose sugar chains will not be added. It can be estimated that the protein was produced.
[0027]
A specific example of a method for losing the function of och1 + is described below. As a linear DNA comprising an och1 + that has lost the ability to produce, for example och1 + linear DNA that about 60% central and replaced with uracil synthesis system gene ura4 + coding region (och1 :: ura4 +) . By introducing it into fission yeast lacking ura4 +, to cause homologous recombination between the och1 + in fission yeast and och1 :: ura4 +. And cultured thereafter in medium without uracil with ura4 +, that is, by och1 + to select the fission yeast replacing a och1 :: ura4 + by homologous recombination, division was lost the function of the och1 + Yeast, ie och1 + disrupted strains can be obtained.
[0028]
The present invention further relates to a method for producing a protein with little glycosylation using an och1 + disrupted strain. Here, the protein may be any protein as long as it is produced by fission yeast, and may be a protein encoded by either a gene derived from fission yeast or a foreign gene not derived from fission yeast. Here, the foreign gene is a gene that is desired to be expressed in fission yeast, and may be a gene derived from any organism such as animal, yeast, or bacteria, but an animal-derived gene is preferable. The foreign gene may have an artificial sequence such as a fusion protein.
[0029]
Examples of gene introduction methods include the spheroplast method, the lithium acetate method, and the electroporation method, but the present invention is not limited thereto. More specifically, for example, in the lithium acetate method, fission yeast in the logarithmic growth phase is kept warm in lithium acetate at 30 ° C. for 1 hour, and then introduced DNA and a polyethylene glycol solution are added, and kept at 30 ° C. for 1 hour. This is heat treated at 43 ° C. for 15 minutes, and then the cells are collected by centrifugation, suspended in a medium, and cultured with shaking at 30 ° C. for 2 hours. When this culture solution is spread on a selective agar medium and cultured at 30 ° C. for 3 days, only yeast cells that have been introduced with the gene and can grow on the selective medium form colonies.
[0030]
The present invention also includes proteins with low glycosylation produced by och1 + disrupted strains. Here, the protein produced from the fission yeast described in the present invention can be purified from the culture supernatant or cells by a standard method such as an affinity column, an ion exchange column, or a gel filtration column. When the introduced gene is designed to encode a fusion protein, for example, in the case of a fusion protein with GST, it is preferable to purify by an affinity column with the fusion protein, and in the case of GST, a glutathione column.
[0031]
The operations described above are described in appropriate manuals such as Sambruck, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. It can be performed according to the method.
[0032]
【Example】
Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
[0033]
[Example 1] Homology of fission yeast och1 + and budding yeast OCH1 consisting of the base sequence shown in SEQ ID NO: 1 Cloned from a genomic DNA library of fission yeast, osmotic pressure was adjusted at high temperatures. A gene och1 + of fission yeast comprising the nucleotide sequence set forth in SEQ ID NO: 1 was found from a genomic DNA fragment capable of complementing properties that can only grow under conditions. As shown in FIG. 1, homology was found between fission yeast och1 + (SpOCH1) and budding yeast OCH1 (ScOCH1).
[0034]
[Example 2] Production of och1 + disruption strain Homologous recombination was caused between DNA containing the nucleotide sequence described in SEQ ID NO: 1 into which mutation was introduced and fission yeast och1 + to produce an och1 + disruption strain.
The DNA comprising the nucleotide sequence of SEQ ID NO: 1, a region interposed between the two HindIII cleavage sites present in och1 + translated region, to produce a substituted DNA to fission yeast ura4 + (Figure 2). Thereafter, the fission yeast by spheroplast method was transformed and cultured in selective medium without uracil with ura4 +, i.e. select the fission yeast with a och1 + which mutation was introduced, och1 + A disrupted strain was created.
[0035]
In Example 3] och1 + form microscopic fission yeast deleted, compared with wild-type fission yeast and fission yeast was deleted och1 +.
As shown in FIG. 3, fission yeast lacking och1 + has a round and short form compared to wild-type fission yeast. Moreover, it is easy to aggregate and the growth rate is slow. Although it exhibits temperature sensitivity that it cannot grow at 37 ° C, it can grow even at 37 ° C under high osmotic pressure conditions by adding about 1.5% sorbitol to the medium.
[0036]
[Example 4] Cultivation of och1 + disrupted strain and analysis of produced protein
We examined whether disruption of och1 + affects the glycosylation of the protein produced.
The och1 + disruption strain and wild-type fission yeast were cultured, and the produced glycoprotein, here, acid phosphatase, was electrophoresed and subjected to activity staining to examine the molecular weight distribution of acid phosphatase.
As a result, as shown in 4, acid phosphatase showed a large smear pattern in the wild type strain (lane 1), whereas the och1 + disrupted strain had a small molecular weight and a sharp band (lane 2). ). This suggests that high-mannose type glycosylation with high diversity and large molecular weight occurs in wild type strains but not in och1 + disrupted strains.
[0037]
Furthermore, in order to investigate whether such a difference in mobility on electrophoresis is due to addition of a sugar chain, a sugar chain cleavage treatment with endoglycosidase H (Endo H) was performed. Since endoglycosidase H is an enzyme that cuts between two N-acetylglucosamine residues at the base of the N-linked sugar chain, the glycoprotein treated with endoglycosidase H has a molecular weight estimated from the polypeptide portion. Shows almost the same mobility.
As a result, the bands of acid phosphatase derived from endoglycosidase H-treated wild-type and och1 + disrupted strains in
[0038]
[Example 5] Mannose transferase activity of och1 + gene product
och1 + mannose transferase activity of the gene product, the Man9GlcNAc2-PA and GDP- mannose fluorescently labeled as a substrate, crushing och1 + disrupted strain, och1 + och1 + disrupted strain was introduced expression vector, and the cells of the wild strain The crude extract was measured as an enzyme.
The fission yeast och1 + gene was incorporated into a pREP1 (Maundrell, K. 1993. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123, 127-130) expression vector to prepare an och1 + expression vector. och1 + crushed och1 + disrupted strain overexpressing the introduced och1 + gene product expression vectors, och1 + disrupted strain was introduced only vector as a control, and the cells of the wild strain, which was precipitated with 100,000 xg Was used as an enzyme. 50 mM Tris HCl (pH 7.5), 10 mM MnCl 2 , 0.6% Triton X-100, 0.5 mM 1-deoxymannojirimycin, 2 mM PA-sugar chain, 1 mM GDP-mannose as the reaction solution Started. After reacting at 30 ° C. for 10, 20, and 30 minutes, the reaction was terminated by treating at 99 ° C. for 5 minutes, and the reaction products were separated by HPLC using a TSK Gel Amide-80 column to analyze the enzyme reaction.
[0039]
As shown in Fig. 5, mannose transfer to Man9GlcNAc2-PA was not observed in the och1 + disrupted strain, but mannose transfer was observed in the wild strain, and the och1 + expression vector was introduced to overexpress the och1 + gene product. More mannose metastasis was observed in the och1 + disrupted strains.
From this result, it was confirmed that the och1 + gene product has activity as mannose transferase activity and is involved in the same reaction as the S. cerevisiae OCH1 gene product.
[0040]
【The invention's effect】
According to the present invention, it is possible to obtain a fission yeast in which the function of glycosyltransferase has been lost, and it is possible to produce a protein with few additional sugar chains even in the fission yeast.
[0041]
[Sequence Listing]
[Brief description of the drawings]
FIG. 1 shows homology between fission yeast och1 + and budding yeast OCH1.
2 is a diagram showing the inserted construct of DNA the och1 + center of ura4 +.
FIG. 3 is a photograph showing the morphology of fission yeast from which och1 + has been deleted.
FIG. 4 is a photograph showing the analysis results of acid phosphatase produced in och1 + disrupted strain.
FIG. 5 shows mannose transferase activity of och1 + gene product.
Claims (9)
(a)配列番号2に記載のアミノ酸配列からなる蛋白質をコードするDNA。
(b)配列番号1に記載の塩基配列からなるDNA。
(c)配列番号1に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、糖転移酵素活性を有する蛋白質をコードする分裂酵母由来のDNA。DNA encoding a glycosyltransferase comprising the DNA according to any one of (a) to (c) below.
(a) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(b) DNA comprising the base sequence set forth in SEQ ID NO: 1.
(c) DNA derived from fission yeast that hybridizes with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions and encodes a protein having glycosyltransferase activity.
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