JP2010142227A - エシェリキア・コリによる異種タンパク質の発酵的製造方法 - Google Patents
エシェリキア・コリによる異種タンパク質の発酵的製造方法 Download PDFInfo
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Abstract
【解決手段】E.コリ菌株を発酵培地中で培養するにあたり、該E.コリ菌株が、異種タンパク質をコードする遺伝子を、シグナルペプチドをコードするシグナル配列と機能的に結合されて含み、かつ該E.コリ菌株が、前記異種タンパク質を発酵培地中に分泌する方法において、該E.コリ菌株が、減衰された(p)ppGpp−シンセターゼII活性を有することを特徴とする方法によって解決される。
【選択図】なし
Description
本発明にとって最も近い先行技術は、relA−突然変異に基づき低減されたPSI−活性を有し、かつrelA−野生型菌株と比較してペリプラズム中に分泌されるタンパク質が培養培地中に増大排出されるE.コリ菌株であり、例えば該菌株は、Gitter他(1995,Appl.Microbiol.Biotechnol.43,89−92)によって記載されている。前記の先行技術に対して減衰されたPSII−活性を有する菌株の利点を明らかにするために、relA−突然変異体を作成した。
E.コリ菌株W3110ΔrelAからのspoT−欠失突然変異体の作成は、原則的に、実施例1に記載した菌株W3110ΔrelAの作成と同様に行った。クロラムフェニコール耐性遺伝子を含み、spoT−遺伝子の5′−領域もしくは3′−領域のそれぞれ50塩基対によってフランキングされている直鎖DNA断片の作成のために、プライマーとしてオリゴヌクレオチドspoTmut−fw(配列番号10)及びspoTmut−rev(配列番号11)と、鋳型としてプラスミドpKD3を使用した。菌株W3110ΔrelA/pKD46を、直鎖DNA断片で形質転換した。W3110ΔrelAの染色体中のspoT−遺伝子とクロラムフェニコール耐性カセットとの効果的な交換は、PCRによって、オリゴヌクレオチドspoT−fw(配列番号12)及びspoT−rev(配列番号13)を使用して、かつ鋳型としてクロラムフェニコール耐性細胞の染色体DNAを使用して確認した。実施例1に記載される手順による染色体からのクロラムフェニコール耐性カセットの除去によって、菌株W3110ΔrelAΔspoTが得られた。
W3110ΔrelAのΔspoT−突然変異体の代わりに、spoT1−アレル(配列番号3;Durfee et al.,2008,J.Bacteriol.190,2597−606)を有するW3110ΔrelAの突然変異体を作成した。このために、spoT1−アレルを、菌株K10(Hfr PO2A tonA22 ompF626 relA1 pit−10 spoT1 T2(R);CGSC番号4234)から、ファージP1を使用した形質導入によって菌株W3110ΔrelAへと移動させた。このために、まず、クロラムフェニコール耐性カセットを、K10の染色体中に、遺伝子recGとgltSとの間でspoT−遺伝子の約2900塩基対を超えて組み込んだ(recG−gltS::cat)。その際、原則的に実施例1に記載されるように行った。クロラムフェニコール耐性カセットを含み、recG−gltSの遺伝子間領域の50塩基対によってそれぞれフランキングされている直鎖DNA断片を、PCRによって、プライマーとしてオリゴヌクレオチドrecG−catfw(配列番号14)及びgltS−catrev(配列番号15)を使用し、かつ鋳型としてプラスミドpKD3を使用して作成し、引き続き該菌株K10/pKD46中に形質転換した。こうして、染色体中に遺伝子recGとgltSとの間で耐性カセットが組み込まれているクロラムフェニコール耐性クローンが得られた(K10recG−gltS::cat)。組み込みが染色体の正しい位置で行われたことは、PCRによって、オリゴヌクレオチドrecG−fw(配列番号16)及びgltS−rev(配列番号17)を使用し、かつ鋳型としてクロラムフェニコール耐性細胞の染色体DNAを使用して確認した。菌株K10recG−gltS::catから、先行技術に従ってP1−溶解産物を作成し、次いでそれを菌株W3110ΔrelAに感染させた。こうして、W3110ΔrelAのクロラムフェニコール耐性形質導入物を得た。形質導入過程では、ほぼ100%の理論的同時形質導入率で、耐性カセットの他にも、染色体上で狭い空間的近隣に局在されるspoT1−アレルが、K10recG−gltS::catからW3110ΔrelAへと移動されるべきであった。実際にそのような場合であることは、spoT−遺伝子のPCRにより、オリゴヌクレオチドspoT−fw(配列番号12)及びspoT−rev(配列番号13)を使用し、かつ鋳型として該菌株の染色体DNAを用いて増幅させ、引き続きPCR産物の配列決定により確認した。実施例1に記載される手順による菌株W3110ΔrelAspoT1recG−gltS::catの染色体からの耐性カセットの除去によって、最終的に、菌株W3110ΔrelAspoT1が得られた。
lpp3−突然変異の導入:
菌株W3110ΔrelAΔspoTもしくはW3110ΔrelAspoT1の染色体におけるlpp野生型遺伝子とlpp3−アレルとの交換は、相同組み換えによって実施した。菌株W3110ΔrelAΔspoTlpp3もしくはW3110ΔrelAspoT1lpp3の作成のために、US2008254511号の実施例3に記載されるのと同じように行った。こうして得られた菌株を、W3110ΔrelAΔspoTlpp3もしくはW3110ΔrelAspoT1lpp3と呼称した。
菌株W3110ΔrelAΔspoTもしくはW3110ΔrelAspoT1の染色体中へのlpp−欠失の導入は、λ−Red−レコンビナーゼ−系を用いて、Datsenko及びWannerの方法(2000,Proc.Natl.Acad.Sci.USA..97:6640−5)によって、かつUS2008254511号の実施例1と同様に実施した。こうして得られた菌株を、W3110ΔrelAΔspoTΔlppもしくはW3110ΔrelAspoT1Δlppと呼称した。
10l−規模でのクレブシエラ・ニューモニアM5a1からのシクロデキストリン−グリコシルトランスフェラーゼ(CGTアーゼ)の産生のために、菌株W3110ΔrelA、W3110spoT1、W3110ΔrelAΔspoT、W3110ΔrelAspoT1、W3110ΔrelAΔspoTΔlpp、W3110ΔrelAΔspoTlpp3、W3110ΔrelAspoT1Δlpp及びW3110ΔrelAspoT1lpp3をCaCl2法によってプラスミドpCGTを用いて形質転換した。プラスミド含有細胞の選択は、アンピシリン(100mg/l)によって行った。
テストバッファー: 5mMのトリスHClバッファー >pH6.5、5mMのCaSO4・2H2O
基質: テストバッファー(pH6.5)中の10%のNoredux溶液
テストバッチ: 1mlの基質溶液+1mlの遠心分離され、場合により希釈された培養上清(5分、12000rpm)+3mlのメタノール
反応温度: 40℃
・ 溶液を予熱する(約5分、40℃で)
・ 酵素溶液を基質溶液に添加する;迅速に混合する(渦ミキサ(Whirl−Mixer))
・ 40℃で3分間インキュベートする
・ 酵素反応をメタノールの添加によって停止させる;迅速に混合する(渦ミキサ)
・ バッチを氷上で冷却する(約5分間)
・ 遠心分離(5分、12000rpm)をし、そして澄明な上清をピペットで取り出す
・ 生じたCDをHPLCによって分析する
酵素活性: A=G*V1*V2/(t*MG)(ユニット/ml)
A=活性
G=CDの含有率(mg/l)=テストバッチ: 単位表面×104/標準溶液(10mg/ml)/単位表面
V1=テストバッチにおける希釈係数(前記のように実施した場合:V1=5)
V2=テストで使用する前の培養上清の希釈係数;非希釈の場合:V2=1
t=反応時間(分)
MG=分子量(g/モル)(CD=973g/モル)
1ユニット=1モルの産物/分
この実施例においては、N末端アミノ酸配列Ala−Thr−Tyr−Thr−Aspを有するヒルジン誘導体の発酵的製造を記載する。ヒルジン誘導体の産生のために、菌株W3110ΔrelA、W3110spoT1、W3110ΔrelAΔspoT、W3110ΔrelAspoT1、W3110ΔrelAΔspoTΔlpp、W3110ΔrelAΔspoTlpp3、W3110ΔrelAspoT1Δlpp及びW3110ΔrelAspoT1lpp3を、まず、それぞれプラスミドpCMT203でCaCl2法によって形質転換した。プラスミド含有細胞の選択は、アンピシリン(100mg/l)によって行った。
インターフェロンα2bの製造のために、菌株W3110ΔrelA、W3110spoT1、W3110ΔrelAΔspoT、W3110ΔrelAspoT1、W3110ΔrelAΔspoTΔlpp、W3110ΔrelAΔspoTlpp3、W3110ΔrelAspoT1Δlpp及びW3110ΔrelAspoT1lpp3を、それぞれプラスミドpIFNでCaCl2法によって形質転換した。プラスミド含有細胞の選択は、アンピシリン(100mg/l)によって行った。
1μlの上清を、サンプルバッファーと混合した(2×Tris SDS−サンプルバッファー(Invitrogen カタログ番号LC2676):0.125Mのトリス塩酸(pH6.8)、4%(w/v)のSDS、20%(v/v)のグリセリン、0.005%(v/v)のブロモフェノールブルー、5%のβ−メルカプトエタノール)。更に、定義された量のインターフェロンα2bを、スタンダードとして一緒に施与した。タンパク質の変性を、100℃に5分間加熱し、氷上で2分間冷却することにより行い、そして遠心分離した。それらのタンパク質を、電気泳動によって、12%のNuPAGE(登録商標)Bis−Tris−Gel(Invitrogen カタログ番号NP0341)中で、1×MES含有ランニングバッファー(Invitrogen カタログ番号NP0002)を用いて分離させた(電気泳動パラメータ: 200Vで40分間)。
湿式ブロッティング法での転写:
モジュール: Amersham: Hoefer TE 22 Mini Tank Transfer Unit、コード番号:80−6204−26
メンブレン: ニトロセルロースメンブレン(Schleicher&Schuell,BA85,硝酸セルロース(E),0.45μmの細孔サイズ)
Whatmanフィルタとニトロセルロースメンブレンを、適切な大きさに切断し、そして発泡物品(スポンジ)で転写バッファー(Invitrogen カタログ番号LC3675)に気泡なく染み込ませた。
積層の構成: 黒い格子、カソードとの接続、各3mm厚を有する2つのスポンジ、Whatmanペーパー、SDSポリアクリルアミドゲル、NCメンブレン、Whatman、6mm厚を有する1つのスポンジ、白い格子、アノードとの接続
転写条件: I=200mAの一定の電流、U=無制限、運転時間60分
25mlのプレハイブリダイゼーションバッファー中で該メンブレンをインキュベートする。
室温で30分間振り動かす。
25mlのプレハイブリダイゼーションバッファー+0.15μg/ml(→3.75μg)の抗ヒトIFN抗体(Pepro Tech EC,Biozolを経由した カタログ番号:500−P32A)中で該メンブレンをインキュベートする。
室温で90分間又は一晩振り動かす。
1×PBSと一緒に室温で10秒間振り動かし、バッファーを捨てる。
1×PBSと一緒に室温で15分間2回振り動かし、バッファーを捨てる。
25mlのプレハイブリダイゼーションバッファー+25μl(1:1000)のヤギ抗ウサギIgGセイヨウワサビペルオキシダーゼコンジュゲート(HRP)(Southern Biotech、Biozolを経由した カタログ番号4050−05)中で該メンブレンをインキュベートする。
室温で60分間振り動かす。
1×PBSと一緒に室温で10秒間振り動かし、バッファーを捨てる。
1×PBSと一緒に室温で15分間2回振り動かし、バッファーを捨てる。
Lumi−Lightウェスタンブロッティング基質(Roche,カタログ番号2015200)を準備する: Lumi−Lightルミノール/エンハンサー溶液とLumi−Light安定ペルオキシド溶液とを1:1の比率で混合する: ニトロセルロースメンブレン当たり3ml。
プレハイブリダイゼーションバッファー: 1×PBS中の5%脱脂粉乳
10×PBS: 100mMのNaH2PO4、1.5MのNaCl、NaOHでpH7.5、0.5%のTriton 100
1×PBS: 10×PBSを完全脱塩水で1:10希釈したもの
定量的評価は、Biorad社製のGS−800 Calibrated Densitometerでイムノブロットをスキャンすることによって、Quantity One 1−D分析ソフト(Biorad)を用いて、施与されたスタンダードと比較することによって実施した。
本実施例は、十分に特徴付けられている抗リゾチーム抗体D1.3のFab−フラグメントの産生を記載する。
本実施例は、抗組織因子(αTF)IgG1−抗体の産生を記載している。
Claims (10)
- E.コリ菌株を発酵培地中で培養することで、異種タンパク質を製造する方法であって、該E.コリ菌株が、異種タンパク質をコードする遺伝子を、シグナルペプチドをコードするシグナル配列と機能的に結合されて含み、かつ該E.コリ菌株が、前記異種タンパク質を発酵培地中に分泌し、該異種タンパク質が発酵培地から分離される、異種タンパク質の製造方法において、前記E.コリ菌株が、減衰された(p)ppGpp−シンセターゼII活性(PSII−活性)を有することを特徴とする方法。
- E.コリ菌株が、そのPSII−活性の改変前の菌株のPSII−活性の最大で50%、好ましくは最大で20%を有することを特徴とする、請求項1に記載の方法。
- E.コリ菌株が、spoT1−突然変異もしくは減衰されたPSII−活性に導くspoT−突然変異を有することを特徴とする、請求項1又は2に記載の方法。
- E.コリ菌株が、PSII−活性の完全な損失をもたらすspoT−突然変異を有することを特徴とする、請求項1から3までのいずれか1項に記載の方法。
- E.コリ菌株が、付加的に、緩和された表現型に導くrelA−遺伝子中の突然変異を有することを特徴とする、請求項1から4までのいずれか1項に記載の方法。
- relA−突然変異が、PSI−活性の完全な損失をもたらすことを特徴とする、請求項5に記載の方法。
- E.コリ菌株が、omp−遺伝子、tol−遺伝子、excD−遺伝子、excC−遺伝子、lpp−遺伝子、pal−遺伝子、env−遺伝子及びleaky−遺伝子の群から選択される遺伝子中に"漏出"突然変異を有することを特徴とする、請求項1から6までのいずれか1項に記載の方法。
- "漏出"突然変異が、lpp−遺伝子中の突然変異、特に有利にはlpp1−突然変異、lpp3−突然変異及びlpp−欠失突然変異の群から選択される突然変異であることを特徴とする、請求項7に記載の方法。
- 異種タンパク質が、真核性タンパク質であることを特徴とする、請求項1から8までのいずれか1項に記載の方法。
- シグナル配列が、E.コリのphoA−遺伝子もしくはompA−遺伝子のシグナル配列又はクレブシエラ・ニューモニアM5a1由来のシクロデキストリン−グリコシルトランスフェラーゼ(CGTアーゼ)のためのシグナル配列又は前記のシグナル配列から誘導される配列番号5の配列であることを特徴とする、請求項1から9までのいずれか1項に記載の方法。
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JP2022033832A (ja) * | 2015-10-30 | 2022-03-02 | シンロジック オペレーティング カンパニー インコーポレイテッド | 消化管炎症低下および/または消化管粘膜バリア強化の利益を享受する疾患処置のために操作された細菌 |
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JP6473460B2 (ja) * | 2014-03-03 | 2019-02-20 | スカラブ ゲノミクス, エルエルシー | 大腸菌における組換えcrm197の産生強化 |
EP3194559A1 (de) | 2015-11-27 | 2017-07-26 | Technische Universität Ilmenau | Verfahren und anordnung zur fermentation |
EP3239291A1 (en) * | 2016-04-26 | 2017-11-01 | Universität Stuttgart | Bacterial strain and method for high throughput of sugar in the microbial conversion into biosynthetic products |
JP7185006B2 (ja) * | 2018-07-06 | 2022-12-06 | ワッカー ケミー アクチエンゲゼルシャフト | 発酵法で組換えタンパク質を放出する細菌株 |
US20240327849A1 (en) * | 2019-08-05 | 2024-10-03 | Wacker Chemie Ag | Bacterial strain for releasing a recombinant protein in a fermentation method |
CN110592109B (zh) * | 2019-08-28 | 2020-10-09 | 黑龙江伊品生物科技有限公司 | 一种spoT基因改造的重组菌株及其构建方法与应用 |
BR112022002535A2 (pt) * | 2019-08-28 | 2022-05-03 | Heilongjiang Eppen Biotech Co Ltd | Sequência nucleotídica, proteína recombinante, vetor recombinante, cepa recombinante, método de construção para a cepa recombinante, e, uso de uma sequência nucleotídica, de uma proteína recombinante, de um vetor recombinante ou de uma cepa recombinante |
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JP2022033832A (ja) * | 2015-10-30 | 2022-03-02 | シンロジック オペレーティング カンパニー インコーポレイテッド | 消化管炎症低下および/または消化管粘膜バリア強化の利益を享受する疾患処置のために操作された細菌 |
JP2021531784A (ja) * | 2018-07-24 | 2021-11-25 | ワッカー ケミー アクチエンゲゼルシャフトWacker Chemie AG | 新規細菌lpp突然変異体及び組換えタンパク質の分泌産生のためのその使用 |
JP7242829B2 (ja) | 2018-07-24 | 2023-03-20 | ワッカー ケミー アクチエンゲゼルシャフト | 新規細菌lpp突然変異体及び組換えタンパク質の分泌産生のためのその使用 |
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DK2204441T3 (da) | 2011-09-12 |
DE102008063900A1 (de) | 2010-06-24 |
EP2204441A1 (de) | 2010-07-07 |
JP5378191B2 (ja) | 2013-12-25 |
EP2204441B1 (de) | 2011-07-13 |
US8053211B2 (en) | 2011-11-08 |
US20100221776A1 (en) | 2010-09-02 |
ATE516341T1 (de) | 2011-07-15 |
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