JP6300121B2 - 大腸菌細胞の高濃度培養方法 - Google Patents
大腸菌細胞の高濃度培養方法 Download PDFInfo
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Description
免疫グロブリンFcフラグメントを発現させる形質変換大腸菌、HM11201(KCCM-10660P)を代表菌株として選別し高濃度培養及び高収率の発現を達成するためにpH-スタット流加発酵に従う段階的方式で、注入培地方法を最適化した。
形質転換大腸菌を初代培養培地(20g/lのトリプトン 、10g/lの酵母抽出物、10g/lのNaCl、207.5g/lのKH2PO4 、50g/lの(NH4)2HPO4、pH6.7)に37℃で空気供給速度1vvmの条件により回分式で培養した。次に、一次注入培地(300〜400g/lの酵母抽出物及び700〜800g/lのブドウ糖)とトレースメタル溶液(268g/lのクエン酸、270g/lのFeCl2 、30g/lのH3BO3 、100g/lのMnCl2、15g/lのCuCl2、25g/lのNa2MoO4 、20g/lのZnCl2 及び0.5mM EDTA)を添加し、流加式で培養した。
上記の実施例1−1で培養した形質転換大腸菌、HM11201によるタンパク質発現の最適条件を確立するために、多様な組成物を二次注入培地として用い、多様な速度で注入した。
Claims (18)
- (i)細胞増殖用培地を用いて、培地のpHが0.1以上上昇するまで大腸菌細胞を回分式で培養する段階:
(ii)細胞培養培地のpHが上昇するとき一次注入培地を添加し大腸菌細胞をpHスタット流加式で培養する段階:及び
(iii)細胞培養培地の600nmでの吸光度(OD600)が120〜180に上昇するときから二次注入培地を添加し大腸菌細胞を流加式で培養する段階であって、培養中に培地のpHが二次注入培地の注入率及び撹拌速度を減少させることによって上昇する、段階を含み、
ステップ(iii)の間、組換えタンパク質の発現を誘導し、組換えタンパク質の発現は、細胞培養培地のOD600が150以上となった時にのみステップ(iii)においてのみ誘導される、大腸菌細胞の培養方法。 - 前記大腸菌は、組換えタンパク質を発現する形質変換された菌株である請求項1に記載の方法。
- 前記形質転換大腸菌株は、HM11201(KCCM-10660P)である請求項2に記載の方法。
- 前記大腸菌が、免疫グロブリンFcフラグメントの組換えタンパク質を発現する形質転換された菌株である、請求項2に記載の方法。
- 前記形質転換大腸菌株は、Lacオペレーターが導入された発現ベクターを含む請求項2に記載の方法。
- イソプロピルβ-D-1-チオガラクトピラノシド(Isopropylβ-D-1-thiogalactopyranoside、IPTG)を添加して組換えタンパク質の発現を誘導する請求項1に記載の方法。
- 前記イソプロピルβ-D-1-チオガラクトピラノシドの添加量は、0.1〜0.5mMである請求項6に記載の方法。
- 前記イソプロピルβ-D-1-チオガラクトピラノシドは、培地の吸光度(OD600)が150以上を示す時だけ添加する請求項6に記載の方法。
- 前記ステップ(ii)は、一次注入培地の注入率は200〜400ml/hrで攪拌速度は400〜800rpmである請求項1に記載の方法。
- 前記注入率及び攪拌速度は、段階的に増加させる請求項9に記載の方法。
- 前記ステップ(ii)は、一次注入培地の注入率及び攪拌速度を3ステップにかけて増加させることで行い、ここで、まず、一次注入培地の注入率は200〜300ml/hrで攪拌速度は400〜600rpmで、それから、注入率は250〜350ml/hrで攪拌速度は500〜700rpmで、最後に、注入率は300〜400ml/hrで攪拌速度は600〜800rpmである請求項9に記載の方法。
- 前記ステップ(iii)において、二次注入培地の注入率は200〜400ml/hrで攪拌速度は400〜800rpmである請求項1に記載の方法。
- 前記注入率及び攪拌速度は、段階的に減少させる請求項12に記載の方法。
- 前記ステップ(iii)は、二次注入培地の注入率及び攪拌速度を3ステップにかけて減少させることで行い、ここで、まず、二次注入培地の注入率は300〜400ml/hrで攪拌速度は600〜800rpmで、それから、注入率は250〜350ml/hrで攪拌速度は500〜700rpmで、最後に、注入率は200〜300ml/hrで攪拌速度は400〜600rpmである請求項12に記載の方法。
- 細胞増殖用培地は、トリプトン、酵母抽出物、NaCl、KH2PO4、(NH4)2HPO4を含む初代増殖培地に、クエン酸、FeCl2、H3BO3、MnCl2、CuCl2、Na2 MoO4、CoCl2、ZnCl2、EDTAを含むトレースメタル溶液(Trace metal solution)が添加された請求項1に記載の方法。
- 一次注入培地は、200〜400g/lの酵母抽出物及び700〜800g/lのブドウ糖を含む請求項1に記載の方法。
- 二次注入培地は、200〜400g/lの酵母抽出物及び500〜700g/lのブドウ糖を含む請求項1に記載の方法。
- 細胞培地の最終吸光度(OD600)が、200以上示す請求項1に記載の方法。
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PCT/KR2013/001980 WO2013137622A1 (en) | 2012-03-12 | 2013-03-12 | Method of culturing e. coli cells for high density |
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JP2015509734A JP2015509734A (ja) | 2015-04-02 |
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US (1) | US9394560B2 (ja) |
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US8883442B2 (en) | 2011-07-13 | 2014-11-11 | Foodchek Systems, Inc. | Culture medium, method for culturing Listeria, and method for detecting Listeria |
WO2015178466A1 (ja) * | 2014-05-21 | 2015-11-26 | 味の素株式会社 | フィブロイン様タンパク質の製造法 |
CN110305831A (zh) * | 2019-07-10 | 2019-10-08 | 鲁东大学 | 一种利用粉丝废水生产渔用生长激素蛋白的方法及其应用 |
CN115011496A (zh) * | 2021-03-04 | 2022-09-06 | 华东理工大学 | 高密度培养海洋病原菌大菱鲆弧菌的培养基及培养方法 |
CN115786299A (zh) * | 2022-11-21 | 2023-03-14 | 福州大学 | 逆转录酶大规模生产时降低包涵体的方法 |
CN116267600B (zh) * | 2022-12-02 | 2024-02-27 | 福建省中科生物股份有限公司 | 一种天门冬属植物的组培苗更新扶壮的培养基及其应用 |
CN115786214B (zh) * | 2022-12-26 | 2023-08-18 | 湖北擎科生物科技有限公司 | 感受态大肠杆菌的高密度发酵培养基及高密度发酵培养方法 |
CN117683699A (zh) * | 2023-12-11 | 2024-03-12 | 广州高腾生物技术有限公司 | 重组大肠杆菌摇瓶培养基及其提高目的蛋白表达量的方法 |
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AR005035A1 (es) | 1995-12-11 | 1999-04-07 | Merck Patent Ges Mit Beschränkter Haftung | Procedimiento para preparar proteinas recombinantes en e. coli, mediante fermentacion con gran concentracion de celulas. |
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GB0522303D0 (en) | 2005-11-01 | 2005-12-07 | Chiron Srl | Culture method |
WO2008013943A2 (en) * | 2006-07-27 | 2008-01-31 | Wyeth | High-cell density fed-batch fermentation process for producing recombinant protein |
CN101613681B (zh) * | 2008-06-24 | 2013-07-10 | 中国科学院上海生命科学研究院 | 重组极端耐热α-淀粉酶的复性与纯化方法 |
CN101818165A (zh) * | 2010-01-19 | 2010-09-01 | 福建省麦丹生物集团有限公司 | 利用vgb基因构建抗贫氧的高密度发酵L-Phe工程质粒的方法 |
CN101831414B (zh) * | 2010-05-25 | 2013-05-22 | 江南大学 | 一种胞外生产重组α-环糊精葡萄糖基转移酶的生产方法 |
CN101921818B (zh) * | 2010-07-15 | 2013-04-10 | 大连理工大学 | 一种生产重组蛋白a的方法 |
KR101152878B1 (ko) | 2011-04-15 | 2012-06-12 | 서울대학교산학협력단 | 글루코오스-6-포스페이트-데하이드로지나아제 및 구아노신-이노신 키나아제의 추가 발현에 의한 재조합 대장균에서 gdp-푸코즈의 생산방법 |
CN102367437A (zh) * | 2011-10-25 | 2012-03-07 | 江南大学 | 一种重组大肠杆菌发酵生产果胶酶的方法 |
KR102041412B1 (ko) * | 2011-12-30 | 2019-11-11 | 한미사이언스 주식회사 | 면역글로불린 Fc 단편 유도체 |
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US9394560B2 (en) | 2016-07-19 |
CN108410788A (zh) | 2018-08-17 |
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