JP2012508001A - 機能グループii莢膜遺伝子クラスターを有しないe.colibl21株 - Google Patents
機能グループii莢膜遺伝子クラスターを有しないe.colibl21株 Download PDFInfo
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- JP2012508001A JP2012508001A JP2011535134A JP2011535134A JP2012508001A JP 2012508001 A JP2012508001 A JP 2012508001A JP 2011535134 A JP2011535134 A JP 2011535134A JP 2011535134 A JP2011535134 A JP 2011535134A JP 2012508001 A JP2012508001 A JP 2012508001A
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Abstract
Description
a.選択マーカーによってグループII莢膜遺伝子クラスターを置換する工程、
b.選択マーカーを除去する工程、
c.PCR分析によって欠失を確認する工程
を含む方法によってグループII莢膜遺伝子クラスターが欠失される、E.coli B株の製造方法も企図する。
株のグリセロールストックを調製するために、適当な抗生物質を含む2mlのLB培地を含む12ml試験管に1つのコロニー移して、220rpmで振盪しながら2時間にわたって37℃で増殖させた。
PCR増幅のためのDNAの起源として、E.coli B BL21(DE3)株を使用した。オリゴヌクレオチドプライマー(LA_BL_GII_F/LA_BL_GII_R)(表3)を合成して、SacII/EcoRIクローニング部位を含むKpsMの5’末端の上流の1001bp領域を標的とした。左アーム(LA)として、ゲノムDNAから当該領域を増幅した。右アーム(RA)に関しては、オリゴヌクレオチドプライマー(RA1_BL_GII_F/RA1_BL_GII_R)(表3)を合成して、BamHI/SacIクローニング部位を含むKpsFの5’末端の上流の1108bp領域を標的とした。左アーム及び右アームのPCR産物を産生した。以下のパラメータ:94℃で50秒間の変性、57℃で60秒間のアニーリング、及び72℃で60秒間のプライマー伸長を全部で30サイクル含むプログラムを使用して、サーマルサイクラーにおいてDNA増幅反応を実施した。正確なバンドサイズのPCR増幅産物を1%アガロースゲル上でゲル電気泳動によって観察して、50μlの増幅産物をゲルから抽出して、置換断片を構築するために使用した。オリゴヌクレオチド配列のリストを表3に示す。
LB培地で一晩にわたって37℃においてE.coli BL21(DE3)を増殖させ、BL21(DE3)のヒートショックコンピテント細胞を標準的な方法によって調製した。プラスミドpKD46でBL21(DE3)コンピテント細胞を形質転換して、形質転換した細胞を0.4OD600nmまで100μg/mlのアンピシリンを添加したLB培地中で増殖させた。Redリコンビナーゼの発現は、L−アラビノース(終濃度1mmol/l)によって1時間にわたって誘導した。
凍結ストックからのE.coli BL21(DE3)Δ(kpsM−kpsF)::cat及びBL21(DE3)ΔdadXΔalr(kpsM−kpsF)::cat細胞を、LB培地において、37℃で、25μg/mlのクロラムフェニコール又は第二の株には25μg/mlのクロラムフェニコールに100mMのD−アラニンとともに増殖させた。2つのノックアウト株のエレクトロポレーションコンピテント細胞を標準的な方法によって調製した。プラスミドpCP20(CmR、AmpR)で2つのノックアウトコンピテント株を形質転換した。薬剤耐性マーカーの除去は、穏やかに振盪しながら1時間にわたって30℃においてSOCブロスで形質転換した細胞を増殖させることによって実施した。プレート状のアンピシリン耐性候補クローンの複数のシングルコロニーを、30℃で一晩培養した後に選択した。候補クローンを、LB培地のみ又はD−アラニンを添加したLB培地で37℃において3時間にわたって増殖させ、プラスミドpCP20を除去した。LB培地のみ又はD−アラニンを添加したLB培地で現れた、5つのシングルコロニーをPCRスクリーニングによってさらに特性決定した。
凍結ストックからのE.coli B BL21(DE3)及びB BL21(DE3)Δ(kpsM−kpsF)細胞を、LBプレートで一晩にわたって37℃で増殖させた。一晩たったプレートから回収した適当量の細胞を、25mlのLB培地を含むバッフル付き250ml振盪フラスコ(baffled shake flask)に約0.05 OD600の濃度まで植菌した。220rpmで振盪しながら8時間にわたって、細胞を37℃で増殖させた。各株のサンプルを1/2時間毎に回収し、OD600で細胞の増殖を比較した。
凍結ストックからのE.coli B BL21(DE3)Δ(kpsM−kpsF)細胞をLBプレート上で増殖させた。ノックアウト株のヒートショックコンピテント細胞を標準的な方法によって調製した。hGHの発現カセットを含むプラスミドpNNC1−g(pET11d−MEAE−hGH(AmpR))で、B BL21(DE3)Δ(kpsM−kpsF)コンピテント細胞を形質転換し、そこから形質転換した細胞のグリセロールストックを調製した。
凍結ストックからのE.coli B BL21(DE3)ΔdadXΔalrΔ(kpsM−kpsF)細胞を、D−アラニンを添加したLB培地で増殖させた。ノックアウト株のヒートショックコンピテント細胞を標準的な方法によって調製した。D−アラニン補足遺伝子を含むpNNC1−1h(pET11d−MEAE−hGH及びalrP−alrCD(AmpR))をAatII/PsiIを使用して消化し、その後の平滑末端及びプラスミドのセルフライゲーションによって、プラスミド上のalr遺伝子の選択のためのbla−プラスミドを得た。プラスミドpNNC1−1h(pET11d−MEAE−hGH,alrP−alrCD,bla−)で、BL21(DE3)ΔdadXΔalrΔ(kpsM−kpsF)コンピテント細胞を形質転換した。D−アラニンを添加していないLBプレート上に一晩であらわれたコロニーを、グリセロールストックの調製のために単離した。
凍結ストックからのE.coli B BL21(DE3)ΔdadXΔalrΔ(kpsM−kpsF)細胞を、D−アラニンを添加したLB培地で増殖させた。ヒートショック方法を使用する形質転換のために、このトリプルノックアウト株のコンピテント細胞を標準的な方法によって調製した。
アースロバクター ウレアファシエンスのシアリダーゼ(AUS,53.4kDa理論的質量)を、C末端のHisタグとともに大腸菌BL21(DE3)Δ(kpsM−kpsF)で製造した。C末端にHis6タグを有する、アースロバクター ウレアファシエンスのシアリダーゼの遺伝子は、T7プロモーターの制御の下に発現ベクターpET−24a(Novagen)にクローニングした。アースロバクター ウレアファシエンス由来のシアリダーゼ酵素を製造するために、以下のタンパク質配列をコードする合成コドン最適化遺伝子を産生した(SEQ ID NO:12):
MAPTPPNSPTLPPGSFSETNLAADRTAANFFYRIPALTYLGNDVVLAAWDGRPGSAADAPNPNSIVQRRSTDGGKTWGPVQVIAAGHVADASGPRYGYSDPSYIYDAEANKVFAFFVYSKDQGFGGSQFGNDDADRNVISSAVIESSDAGVTWSQPRLITSVTKPGTSKTNPAAGDVRSNFASSGEGIQLKYGPHKGRLIQQYAGDVRQADGSNKIQAYSVYSDDHGVTWHKGANVGDRMDENKTVELSDGRVLLNSRDNANRGYRKVAVSTDGGATYGPVSQDTELPDPANNGAIARMFPNAAQGSADAKKLIFTNANSKTGRENVSARVSCDDGETWPGVRTIRSGFSAYSTVTRLADGKFGVLYEGNYTDNMPFATFDDAWLNYVCAPLAVPAVNIAPSATQEVPVTVTNQEATTLSGATATVYTPSGWSATTVPVPDVAPGASVTVTVALTAPADASGPRSLNAAFTTADGRVSQFTFTATTPVAPQVGLTITGSALEHHHHHH。
ヒトFGF21のDNA及びアミノ酸配列は、例えば、Biochim. Biophys. Acta 1492(1):203−206 (2000)のNishimura et alに開示されている。前記配列は、公開データベースからも得られる(各々、Accession nos. EMBL:AB021975及びUNIPROT:Q9NSA1)。
MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPA-LPEPPGILAPQPPDVGSSDPLSMVGPS QGRSPSYAS。
37アミノ酸のアミロイドペプチドであるアミリンの配列は、Proc. Natl. Acad. Sci. (84) : 8628−8632, (1987)のCooper et al.によって公開された。遺伝子をクローニングして、サーモトガ・マリティマ(thermotoga maritima)由来のリボソームタンパク質RL23にN末端に融合した。146アミノ酸の融合タンパク質は、以下の配列(SEQ ID NO:14)を有する:
KQEKLSLHDVLIRPIITEKALILREQRKYVFEVNPLANKNLVKEAVEKLFNVKVEKVNILNMKPKPKRRGIFEGKTRSWKKAVVTLKEGYTIKELEGEHSSSSDDDDKKCNTATCATQRLA-NFLVHSSNNFGAILSSTNVGSNTY。
146アミノ酸長のヒトレプチンの配列は、Diabetes (44): 855−858 (1995)のMasuzaki et alに公開されている。前記タンパク質は、21アミノ酸のシグナルペプチドが添加されてヒトにおいて分泌される。E.coliで細胞内に発現させた成熟レプチンは、以下の配列(SEQ ID NO:15)を有する:
MVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。
Claims (15)
- 不可逆的に不活性化されたグループII莢膜遺伝子クラスターを有することを特徴とする、E.coli B BL21株。
- グループII莢膜遺伝子クラスターの全体又は一部の欠失を有することを特徴とする、請求項1に記載のE.coli B BL21株。
- グループII莢膜遺伝子クラスターの全体の欠失を有することを特徴とする、請求項1に記載のE.coli B BL21株。
- 前記E.coliがE.coli B BL21(DE3)である、請求項1から3のいずれか一項に記載のE.coli B BL21株。
- 前記E.coliがE.coli B BL21(DE3)ΔdadXΔalrである、請求項1から3のいずれか一項に記載のE.coli株。
- ペプチドをコードする挿入DNA配列を有する発現ベクターも含む、請求項1から5のいずれか一項に記載のE.coli B BL21株。
- 前記ペプチドが、hGH、グルカゴン様ペプチド、インターロイキン、インスリンアナログ、野生型アジポネクチン、FGF−21、トリプシン、アプロチニン、アミリン及びレプチン並びにそれらのアナログ、並びに酵素、例えば、シアリダーゼ、トランスグルタミナーゼ(tGase)、HRV(ヒトライノウイルス)3Cプロテアーゼ、タバコエッチウイルス(TEV)プロテアーゼ、及びそれらのバリアントからなる群から選択される、請求項1から6のいずれか一項に記載のE.coli B BL21株。
- 前記グループII莢膜遺伝子クラスターが、
a.選択マーカーによってグループII莢膜遺伝子クラスターを置換する工程、
b.前記選択マーカーを除去する工程、
c.PCR分析によって欠失を確認する工程
を含む方法によって欠失される、請求項1から7のいずれか一項に記載のE.coli B株の製造方法。 - 前記選択マーカーが抗生物質耐性遺伝子である、請求項8に記載のE.coli B株の製造方法。
- 前記抗生物質耐性遺伝子が、クロラムフェニコール、テトラサイクリン、アンピシリン、カナマイシン、バンコマイシン、及びエリスロマイシンからなる群から選択される、請求項9に記載のE.coli B株の製造方法。
- 前記抗生物質がクロラムフェニコールである、請求項9に記載のE.coli B株の製造方法。
- 請求項1から7のいずれか一項に記載のE.coli B株を適切な培養培地で培養して、その後に、よく知られた技術によって発現したペプチドを単離及び精製することを含む、ペプチドの製造方法。
- 前記ペプチドがhGHである、請求項12に記載の方法。
- 前記ペプチドがFGF−21である、請求項12に記載の方法。
- 前記ペプチドがアミリンである、請求項12に記載の方法。
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JP2001522347A (ja) * | 1996-06-27 | 2001-11-13 | ヴァージニア テック インテレクチュアル プロパティーズ,インコーポレーテッド | 莢膜被包生物によって引き起こされる疾病用の組換えワクチン |
JP2004528005A (ja) * | 2000-08-15 | 2004-09-16 | ファージ バイオテクノロジー コーポレイション | 生物学的に活性なタンパク質およびペプチドのファージ依存性超産生 |
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