JP2009501789A - 顆粒球コロニー刺激因子(g−csf)の変異型およびその化学的に抱合されたポリペプチド - Google Patents
顆粒球コロニー刺激因子(g−csf)の変異型およびその化学的に抱合されたポリペプチド Download PDFInfo
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- JP2009501789A JP2009501789A JP2008522703A JP2008522703A JP2009501789A JP 2009501789 A JP2009501789 A JP 2009501789A JP 2008522703 A JP2008522703 A JP 2008522703A JP 2008522703 A JP2008522703 A JP 2008522703A JP 2009501789 A JP2009501789 A JP 2009501789A
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Abstract
Description
図1は、PEG−G−CSF抱合反応の模式図である。
A.組み換え体G−CSF(rG−CSF)の調製
本発明で用いられる組み換え体G−CSF(rG−CSF)を、本発明の出願人による韓国特許第230579号に記載される株(KFCC−10961)を用いて発現させた。それゆえ、調製した組み換え体G−CSFはフィルグラスチムのごとく大腸菌に由来し、配列番号2で同定したアミノ酸配列を有する。
システイン導入変異型G−CSFを、韓国特許第230579号に記載されるG−CSF発現株(KFCC−10961)から調製した。G−CSF発現株をLB培養培地中で撹拌させながら12時間培養し、次いでキアゲン(Quiagen)社製のプラスミド調製キットを用いてG−CSF発現ベクター(pGW2)を単離した。表1にリストされるシステイン導入遺伝子(30−40塩基対の大きさの相補的ポリヌクレオチド二本鎖)を含む相補的変異プライマーを用いて鋳型として単離したG−CSF発現ベクター(pGW2)上においてポリメラーゼ鎖反応(PCR)を行った。次いで、PCRでクローン化したG−CSF発現ベクターをDPN1酵素で処理して鋳型を取り除き、続いてルビジウムにより処理したMC 1061大腸菌に形質導入した。形質導入株をスクリーニングするために形質導入した宿主をアンピシリン培養培地で培養した。変異と鋳型除去のためのPCR産物の酵素処理を、部位特異的変異キット(ストラタジーン社のQuickChange Site-Directed Mutagenesis Kit)で利用可能なpfuTurboDNAポリメラーゼとDPN1酵素各々を用いて使用説明書に従って行った。変異をプライマー(5’−GCGGATCCTGCCTGA−3’)を用いるPCRによる塩基配列決定の解析により確認した。
システイン導入変異型G−CSF発現株を5μg/ml アンピシリンを添加した5ml LB培養培地で12時間培養し、次いで5μg/ml アンピシリンを添加した500ml LB培地に移して撹拌しながら培養した。この株の吸光度(OD)が0.5に達したら、最終濃度1%(w/v)でアラビノースを各培養培地に添加し、約7時間撹拌しながら培養してシステイン導入変異型G−CSFを過剰発現させた。
過剰発現させたシステイン導入変異型G−CSF発現株を遠心分離し、Tris 20mMと150mM NaClで懸濁し、懸濁した株をソニケーターを用いて破壊した。破壊した株を遠心分離し、2%(w/v)のデオキシコール酸ナトリウムで洗浄して封入体に回復させた。システイン導入変異型の封入体をpH8.8の7M 尿素、20mM Tris、および100mM NaClに溶解させ、pH8.8の3.3M 尿素、5mM Tris、2mM 還元型グルタチオン、および0.2M 酸化型グルタチオンで10倍希釈した。リフォールディングの段階を4℃で18時間撹拌しながら行った。
リフォールディングサンプルとしてのシステイン導入変異型G−CSFをHClによる滴定でpH4.5にした後、生じた沈殿物を遠心分離により除去した。活性を有するシステイン導入変異型G−CSFを精製するため、20mM リン酸ナトリウムをpH4.0で平衡化したSP−セファロースファーストフロー(fast flow)カラムに注入し、平衡化緩衝液を用いて洗浄し、塩濃度100〜5000mM NaClの勾配で溶離した。
セクションE−1の記載と同一の方法により単離したシステイン導入変異型G−CSFのSP−セファロース流出液サンプルのpHをpH7.5に調整した後、生じた溶液に約20kDaの平均分子量を有するメトキシ−PEG−ビニルスルホンを添加してシステイン導入変異型G−CSF:メトキシ−PEG−マレイミドのモル比が1:5になるようにした。次に、ゆっくり撹拌しながら4℃で約18時間反応させた。
セクションE−1の記載と同一の方法により単離したシステイン導入変異型G−CSFのSP−セファロース流出液サンプルのpHをpH7.0に調整した後、生じた溶液に約20kDaの平均分子量を有するメトキシ−PEG−ヨードアセトアミドを添加してシステイン導入変異型G−CSF:メトキシ−PEG−ヨードアセトアミドのモル比が1:2になるようにした。次に、ゆっくり撹拌しながら4℃の暗室で約48時間反応させた。
調製したペグ化システイン導入変異型G−CSFのpHをpH4.0に調整した後、生じた生成物を緩衝溶液(20mM 第一Na2HPO4、pH4.8)で3倍に希釈し、平衡化緩衝液と同一の緩衝液、すなわち20mM 第一Na2HPO4、pH4.8を含む平衡化緩衝液を用いて平衡化したSP−セファロースファーストフローカラムに注入した。次いで、生じた生成物を緩衝溶液(20mM 第一Na2HPO4、50mM NaCl、pH4.8)で洗浄し、塩濃度50〜500mM NaClの勾配で溶離した。システイン導入変異型G−CSFから未反応のG−CSFを除去するために、SP−セファロースカラムから溶離したペグ化システイン導入変異型G−CSFにサイズ除外クロマトグラフィーを行った。SP−セファロース流出液を濃縮し、緩衝溶液(20mM 第一Na2HPO4、100mM NaCl(pH4.0))で平衡化したSuperdex 200(2.5X50cm、ファルマシア(Pharmacia))に注入し、1ml/分の溶離速度において同一の緩衝溶液で溶離した。単離したペグ化システイン導入変異型G−CSFの精製度をSDS−PAGE解析により調べた。
(表1)
アミノ酸におけるセリンによるシステイン(Cys)の置換とCDループ(Gly126−Ser143)におけるセリンによる位置18のシステインの置換を包含することにより変異型を調製するために、システイン導入変異をCys 18 Ser置換変異型の株を用いて行い、システイン導入変異を上記に記載と同一の方法で行ったことを除いて、対象の変異型を実施例1から11と同一の方法で調製した。本発明者らは、配列番号2で同定されるrG−CSFのCDループに導入されたシステインを有する変異型を調製した。
(表2)
CDループ(Gly126−Ser143)へのシステイン(Cys)の挿入によって変異型を調製するために、表3に記載したシステイン導入遺伝子を含む相補的変異プライマー(30−40塩基対の大きさを有する相補的ポリヌクレオチド二本鎖)を用いて鋳型として単離したG−CSF発現ベクター(pGW2)上でポリメラーゼ鎖反応(PCR)を行ったことを除いて、対象の変異型を実施例1から11と同一の方法で調製した。
(表3)
アミノ酸におけるシステイン(Cys)の挿入およびCDループ(Gly126−Ser143)におけるセリンと位置18システインの置換の両方を包含することにより変異型を調製するために、Cys 18 Serにより置換された変異型株を用いてシステイン導入変異を行い、システイン導入変異を上記の記載と同一の様式で行うことを除いて、実施例23から28と同一の様式で対象の変異型を調製した。
(表4)
実施例1から34で調製したシステイン導入変異型G−CSF PEG抱合体の生物学的活性を測定し、フィルグラスチン(大腸菌由来のG−CSF)の活性と比較した。マウス骨髄性白血病細胞株NFS−60(ATCC X65622)の細胞増殖を介して活性測定を行った。NFS−60細胞を10%(v/v)FBS(ウシ胎児血清)と5% WEHI−1640細胞株培地溶液を含むRPMI1640培地で培養し、10%(v/v)FBSを含むRPMI培地中で懸濁して約2X105細胞/mlの濃度にした。懸濁した細胞懸濁液各50μlを添加して96ウェルマイクロタイタープレートの各ウェルに約1X104細胞を含むようにした。
実験的実施例1で調べた各20kDa PEG抱合システイン導入変異型G−CSF、すなわち、20kDa PEG−M1、20kDa PEG_M1_S、20kDa PEG−M2、および20kDa PEG−M2_Sの血清の保持時間を測定した。各群にについて、フィルグラスチム(コントロール)ならびに実施例で調製した20kDa PEG−M1、20kDa PEG_M1_S、20kDa PEG−M2、および20kDa PEG−M2_Sを各5匹のSDラット(体重200−250g、6週齢のスプラーグドーリーラットの雄)に対して1kg体重あたり100μgで皮下注射し、注射後0.5、1、2、4、8、12、16、20、24、30、36、48、60、72、96、120、144、168時間後に血液サンプルを採取した。血液サンプルを室温で1時間凝血させ、微量遠心機を用いて10000rpmで5分間遠心分離し、それにより細胞を除去した。モノクローナル抗体を用いる酵素結合免疫吸着法(ELISA)により血しょうG−CSFの量を測定した。
(表6)
実験的実施例1で決定した20kDa PEG抱合システイン導入変異型G−CSF各々、すなわち、20kDa PEG−M1、20kDa PEG_M1_S、20kDa PEG−M2、および20kDa PEG−M2_Sの好中球活性を測定した。各群について、フィルグラスチム(コントロール)ならびに実施例で調製した20kDa PEG−M1、20kDa PEG_M1_S、20kDa PEG−M2、および20kDa PEG−M2_Sを各5匹のSDラット(体重200−250g、6週齢のスプラーグドーリーラットの雄)に対して1kg体重あたり100μgで皮下注射し、血液サンプルを経時的に目から採血することによって採取した。次いで、血漿中の好中球数をカウントした。
Claims (12)
- 配列番号3で同定されるアミノ酸配列を含むヒト顆粒球コロニー刺激因子(G−CSF)の変異型であって、配列番号1で同定されるアミノ酸配列を有するG−CSFの位置133のスレオニン(Thr)残基がシステイン(Cys)残基に置換された変異型。
- 配列番号3で同定されるアミノ酸配列を含むヒトG−CSFの抱合された変異型であって、配列番号1で同定されるアミノ酸配列を含むG−CSFの位置133のスレオニン(Thr)残基がシステイン(Cys)残基に置換され、前記置換Cys残基が非タンパク質化合物に結合している変異型。
- 前記変異型が、配列番号4で同定されるアミノ酸配列を含むタンパク質であって、位置17のシステイン(Cys)残基がセリン(Ser)残基に置換された、請求項2の抱合された変異型。
- 非タンパク質化合物が、マレイミド、ビニルスルホン、ヨードアセトアミドおよびオルトピリジルジスルフィドを含む群から選択される1種類と結合する、ポリエチレングリコール(PEG)、ポリビニルアルコール(PVA)、ポリカルボキシル酸およびポリビニルピロリドンを含む群から選択される1種類の化合物である、請求項2または3の抱合された変異型。
- 非タンパク質化合物が、約2から約100kDaまでの範囲の分子量であり、請求項2または3の抱合された変異型。
- PEGが約5ないし約100kDaの範囲の分子量を有する、請求項4の抱合された変異型。
- 配列番号5に含まれるアミノ酸配列を含むヒト顆粒球コロニー刺激因子(G−CSF)の変異型であって、システイン(Cys)残基が、配列番号1に含まれるG−CSFの位置135のグリシン(Gly)残基と位置136のアラニン(Ala)残基の間に挿入される変異型。
- 配列番号5に含まれるアミノ酸配列を含むヒト顆粒球コロニー刺激因子(G−CSF)の抱合された変異型であって、システイン(Cys)残基が配列番号1に含まれるアミノ酸配列を含むG−CSFの位置135のグリシン(Gly)と位置136のアラニン(Ala)残基の間に挿入され、非タンパク質化合物が挿入されたシステイン残基に結合されている変異型。
- 変異型が、位置17のシステイン(Cys)残基がセリン(Ser)残基に置換された配列番号6に含まれるアミノ酸配列を含むタンパク質である、請求項8の抱合された変異型。
- 非タンパク質化合物が、マレイミド、ビニルスルホン、ヨードアセトアミドおよびオルトピリジルジスルフィドを含む群から選択される1種類と結合している、ポリエチレングリコール(PEG)、ポリビニルアルコール(PVA)、ポリカルボキシル酸およびポリビニルピロリドンを含む群から選択される1種類の化合物である、請求項8または9の抱合された変異型。
- 非タンパク質化合物が、約2から約100kDaまでの範囲の分子量を有する、請求項8または9の抱合された変異型。
- PEGが約5から約100kDaまでの範囲の分子量を有する、請求項10の抱合された変異型。
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CN116334057B (zh) * | 2022-08-16 | 2024-07-19 | 北京理工大学 | 一种利用聚乙二醇马来酰亚胺衍生物构建的酶组装体及其制备方法和应用 |
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