JP2015534814A - 組換えクロストリジウムボツリヌス神経毒 - Google Patents
組換えクロストリジウムボツリヌス神経毒 Download PDFInfo
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Abstract
Description
最大10個のフェニルアラニン低速コドンと、
最大18個のチロシン低速コドンと、
最大2個のシステイン低速コドンと、
最大4個のヒスチジン低速コドンと、
最大3個のグルタミン低速コドンと、
最大19個のプロリン低速コドンと、
最大16個のセリン低速コドンと、
最大5個のロイシン低速コドンと、を含む。
最大10個のフェニルアラニン低速コドンと、
最大18個のチロシン低速コドンと、
最大2個のシステイン低速コドンと、
最大4個のヒスチジン低速コドンと、
最大3個のグルタミン低速コドンと、
最大19個のプロリン低速コドンと、
最大16個のセリン低速コドンと、
最大5個のロイシン低速コドンと、
最大10個のアルギニン低速コドンと、を含む。
位置177のグリシン、位置198のセリン、位置340のアラニン、位置773のロイシン、位置963のロイシン、位置964のグルタミン、位置967のアラニン、位置1195のアスパラギン。
(1)ProがP'1位置にあると、通常、トリプシンによる切断に対する感受性が低減される(但し、Trpが位置P2にある時は該当しない)。
(2)Cys又はAspがP2位置にあり且つAspがP'1位置にあると、トリプシンによる切断に対する感受性が低減される。
(3)CysがP2位置にあり且つHis又はTryがP'1位置にあると、トリプシンによる切断に対する感受性が低減される。
(1)ProがP'1位置にあると、通常、トリプシンによる切断に対する感受性が低減される(但し、Met又は恐らくはGluが位置P2にある時は該当しない)。
(2)CysがP2位置にあり且つLysがP'1位置にあると、トリプシンによる切断に対する感受性が低減される。
(3)ArgがP2位置にあり且つHis又はArgがP'1位置にあると、トリプシンによる切断に対する感受性が低減される。
(a)上述した活性2本鎖BoNT/E1タンパク質と、
(b)安定化剤と、を含む、固体又は液体医薬組成物を提供する。
(a)上述した活性2本鎖BoNT/E1タンパク質と、
(b)界面活性剤である非タンパク質安定化剤と
(c)水と、を含む液体医薬組成物であり、
前記液体医薬組成物は、タンパク質を安定化剤として含まず、
前記液体医薬組成物は、トリプシンを実質的に含まない(例えば、前記液体医薬組成物は、BoNT/E1タンパク質100ng当たり100pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり10pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり7pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり5pg未満のトリプシンを含み、好ましくは、前記液体医薬組成物は、BoNT/E1タンパク質100ng当たり10pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり7pg未満のトリプシンを含む)。
(a)上述した活性2本鎖BoNT/E1タンパク質と、
(b)界面活性剤である非タンパク質安定化剤と、
(c)塩化ナトリウムと、
(c)pHを5.5〜7.5に維持する緩衝剤と、
(e)二糖と、
(f)滅菌水と、を含む液体医薬組成物であり、
前記液体医薬組成物は、タンパク質を安定化剤として含まず、
前記液体医薬組成物は、トリプシンを実質的に含まない(例えば、前記液体医薬組成物は、BoNT/E1タンパク質100ng当たり100pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり10pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり7pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり5pg未満のトリプシンを含み、好ましくは、前記液体医薬組成物は、BoNT/E1タンパク質100ng当たり10pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり7pg未満のトリプシンを含む)。
配列番号:2 BoNT/E1アミノ酸配列
配列番号:3 野生型BoNT/E1核酸配列
ATGCCGAAAATCAACTCTTTCAACTACAACGACCCGGTTAACGACCGTACCATCCTGTAT
ATCAAACCGGGTGGTTGCCAGGAGTTCTACAAATCTTTCAACATCATGAAAAACATCTGG
ATCATCCCGGAACGTAACGTTATCGGTACCACCCCGCAGGACTTCCACCCGCCGACCTCT
CTGAAAAACGGTGACTCTTCTTACTACGACCCGAACTACCTCCAGTCTGACGAAGAAAAA
GACCGTTTCCTGAAAATCGTTACCAAAATCTTCAACCGTATCAACAACAACCTGTCTGGT
GGTATCCTGCTGGAAGAACTGTCTAAAGCTAACCCGTACCTGGGTAACGACAACACCCCG
GACAACCAGTTCCACATCGGTGACGCTTCTGCTGTTGAAATCAAATTCTCTAACGGTTCT
CAGGACATCCTGCTGCCGAACGTTATCATCATGGGTGCTGAACCGGACCTGTTCGAAACC
AACTCTTCTAACATCTCTCTGCGTAACAACTACATGCCGTCTAACCACGGTTTCGGTTCT
ATCGCTATCGTTACCTTCTCTCCGGAATACTCTTTCCGTTTCAACGACAACAGCATGAAC
GAGTTCATCCAGGACCCGGCTCTGACCCTGATGCACGAACTGATCCACTCTCTGCACGGT
CTGTACGGTGCTAAAGGTATCACCACCAAATACACCATCACCCAGAAACAGAACCCGCTG
ATCACCAACATCCGTGGTACCAACATCGAAGAGTTCCTGACCTTCGGTGGTACCGACCTG
AACATCATCACCTCTGCTCAGTCTAACGACATCTACACCAACCTGCTGGCTGACTACAAA
AAAATCGCTTCTAAACTGTCTAAAGTTCAGGTTTCTAACCCGCTGCTGAACCCGTACAAA
GACGTTTTCGAAGCTAAATACGGTCTGGACAAAGACGCTTCTGGTATCTACTCTGTTAAC
ATCAACAAATTCAACGACATCTTCAAAAAACTGTACTCTTTCACCGAGTTCGACCTGGCG
ACCAAATTCCAGGTTAAATGCCGTCAGACCTACATCGGTCAGTACAAATACTTCAAACTG
TCTAACCTGCTGAACGACTCTATCTACAACATCTCTGAAGGTTACAACATCAACAACCTG
AAAGTTAACTTCCGTGGTCAGAACGCTAACCTGAACCCGCGTATCATCACCCCGATCACC
GGTCGTGGTCTGGTTAAAAAAATCATCCGTTTCTGCAAGAATATTGTAAGCGTTAAAGGA
ATAAGAAAAAGTATCTGCATCGAAATCAACAACGGTGAACTGTTCTTCGTTGCTTCTGAA
AACTCTTACAACGACGACAACATCAACACCCCGAAAGAAATCGACGACACCGTTACCTCT
AACAACAACTACGAAAACGACCTGGACCAGGTTATCCTGAACTTCAACTCTGAATCTGCT
CCGGGTCTGTCTGACGAAAAACTGAACCTGACCATCCAGAACGACGCTTACATCCCGAAA
TACGACTCTAACGGTACCTCTGACATCGAACAGCACGACGTTAACGAACTGAACGTTTTC
TTCTACCTGGACGCTCAGAAAGTTCCGGAAGGTGAAAACAACGTTAACCTGACCTCTTCT
ATCGACACCGCTCTGCTGGAACAGCCGAAAATCTACACCTTCTTCTCTTCTGAGTTCATC
AACAACGTTAACAAACCGGTTCAGGCTGCTCTGTTCGTTTCTTGGATTCAGCAGGTTCTG
GTTGACTTCACCACCGAAGCTAACCAGAAATCTACCGTTGACAAAATCGCTGACATCTCT
ATCGTTGTTCCGTACATCGGTCTGGCTCTGAACATCGGTAACGAAGCTCAGAAAGGTAAC
TTCAAAGACGCTCTGGAACTGCTGGGTGCTGGTATCCTGCTGGAGTTCGAACCGGAACTG
CTGATCCCGACCATCCTGGTTTTCACCATCAAATCTTTCCTGGGTTCTTCTGACAACAAA
AACAAAGTTATCAAAGCTATCAACAACGCTCTGAAAGAACGTGACGAAAAATGGAAAGAA
GTTTACTCTTTCATCGTTTCTAACTGGATGACCAAAATCAACACCCAGTTCAACAAACGT
AAAGAACAGATGTACCAGGCTCTCCAGAACCAGGTTAACGCTATCAAAACCATCATCGAA
TCTAAATACAACTCTTACACCCTGGAAGAAAAAAACGAACTGACCAACAAATACGACATC
AAACAGATCGAAAACGAACTGAACCAGAAAGTTTCTATCGCTATGAACAACATCGACCGT
TTCCTGACCGAATCTTCTATCTCTTACCTGATGAAACTCATCAACGAAGTTAAAATCAAC
AAACTGCGTGAATACGACGAAAACGTTAAAACCTACCTGCTGAACTACATCATCCAGCAC
GGTTCTATCCTGGGTGAATCTCAGCAGGAACTGAACTCTATGGTTACCGACACCCTGAAC
AACTCTATCCCGTTCAAACTGTCTTCTTACACCGACGACAAAATCCTGATCTCTTACTTC
AACAAATTCTTTAAACGCATTAAGAGTTCATCGGTTCTGAATATGCGGTACAAAAATGAT
AAATATGTCGATACTTCTGGATATGATAGCAATATCAACATTAACGGCGACGTGTATAAA
TATCCGACAAATAAAAACCAGTTTGGGATATATAACGACAAGCTGTCGGAGGTCAATATT
TCTCAAAACGACTATATCATTTACGATAATAAATATAAAAACTTTAGCATTAGTTTTTGG
GTTCGTATACCTAATTATGACAATAAAATTGTAAATGTGAATAACGAGTATACCATTATA
AACTGTATGCGCGACAATAACAGTGGTTGGAAGGTATCGCTGAACCATAATGAGATTATC
TGGACCCTGCAGGATAATGCAGGTATAAACCAGAAACTGGCTTTTAACTATGGAAACGCA
AATGGGATCTCAGATTACATTAATAAATGGATTTTTGTTACCATTACGAACGATCGCTTA
GGCGACTCAAAACTTTATATTAATGGCAATCTGATAGATCAGAAATCAATCTTAAATTTG
GGCAATATTCATGTCTCTGATAACATCTTGTTCAAGATCGTTAATTGCAGTTACACTCGT
TATATTGGCATTCGTTACTTTAATATCTTCGATAAAGAACTGGACGAGACGGAAATCCAG
ACTCTGTATTCAAACGAGCCCAATACTAATATATTGAAAGATTTTTGGGGTAACTATCTT
TTATATGATAAAGAATACTATCTCCTGAATGTATTGAAGCCAAACAATTTCATAGATAGA
CGCAAGGATAGCACATTAAGTATCAACAATATCAGATCTACTATACTGTTAGCAAATCGC
CTCTACTCCGGTATTAAAGTGAAGATTCAGCGGGTTAATAACTCCAGTACCAATGATAAT
CTGGTCCGTAAGAACGATCAGGTATACATCAATTTCGTCGCGAGCAAAACTCATCTCTTC
CCGCTTTACGCCGATACAGCTACGACAAACAAGGAAAAAACCATAAAAATTTCCAGCTCC
GGAAACAGATTCAATCAAGTAGTTGTAATGAACTCTGTGGGTAATAATTGTACGATGAAC
TTTAAGAATAACAATGGGAACAATATTGGACTTTTGGGCTTCAAAGCCGACACAGTGGTG
GCGTCCACCTGGTATTACACGCACATGCGGGACCATACGAATTCGAACGGTTGCTTCTGG
AACTTTATCTCGGAAGAACACGGGTGGCAAGAAAAATAA
MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS
LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP
DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGS
IAIVTFSPEYSFRFNDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL
ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK
DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKL
SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG
IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA
PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS
IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS
IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK
NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE
SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKLINEVKIN
KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF
NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI
SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII
WTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL
GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL
LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN
LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMN
FKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK
ATGCCAAAAATTAATAGTTTTAATTATAATGATCCTGTTAATGATAGAACAATTTTATAT
ATTAAACCAGGCGGTTGTCAAGAATTTTATAAATCATTTAATATTATGAAAAATATTTGG
ATAATTCCAGAGAGAAATGTAATTGGTACAACCCCCCAAGATTTTCATCCGCCTACTTCA
TTAAAAAATGGAGATAGTAGTTATTATGACCCTAATTATTTACAAAGTGATGAAGAAAAG
GATAGATTTTTAAAAATAGTCACAAAAATATTTAATAGAATAAATAATAATCTTTCAGGA
GGGATTTTATTAGAAGAACTGTCAAAAGCTAATCCATATTTAGGGAATGATAATACTCCA
GATAATCAATTCCATATTGGTGATGCATCAGCAGTTGAGATTAAATTCTCAAATGGTAGC
CAAGACATACTATTACCTAATGTTATTATAATGGGAGCAGAGCCTGATTTATTTGAAACT
AACAGTTCCAATATTTCTCTAAGAAATAATTATATGCCAAGCAATCACGGTTTTGGATCA
ATAGCTATAGTAACATTCTCACCTGAATATTCTTTTAGATTTAATGATAATAGTATGAAT
GAATTTATTCAAGATCCTGCTCTTACATTAATGCATGAATTAATACATTCATTACATGGA
CTATATGGGGCTAAAGGGATTACTACAAAGTATACTATAACACAAAAACAAAATCCCCTA
ATAACAAATATAAGAGGTACAAATATTGAAGAATTCTTAACTTTTGGAGGTACTGATTTA
AACATTATTACTAGTGCTCAGTCCAATGATATCTATACTAATCTTCTAGCTGATTATAAA
AAAATAGCGTCTAAACTTAGCAAAGTACAAGTATCTAATCCACTACTTAATCCTTATAAA
GATGTTTTTGAAGCAAAGTATGGATTAGATAAAGATGCTAGCGGAATTTATTCGGTAAAT
ATAAACAAATTTAATGATATTTTTAAAAAATTATACAGCTTTACGGAATTTGATTTAGCA
ACTAAATTTCAAGTTAAATGTAGGCAAACTTATATTGGACAGTATAAATACTTCAAACTT
TCAAACTTGTTAAATGATTCTATTTATAATATATCAGAAGGCTATAATATAAATAATTTA
AAGGTAAATTTTAGAGGACAGAATGCAAATTTAAATCCTAGAATTATTACACCAATTACA
GGTAGAGGACTAGTAAAAAAAATCATTAGATTTTGTAAAAATATTGTTTCTGTAAAAGGC
ATAAGGAAATCAATATGTATCGAAATAAATAATGGTGAGTTATTTTTTGTGGCTTCCGAG
AATAGTTATAATGATGATAATATAAATACTCCTAAAGAAATTGACGATACAGTAACTTCA
AATAATAATTATGAAAATGATTTAGATCAGGTTATTTTAAATTTTAATAGTGAATCAGCA
CCTGGACTTTCAGATGAAAAATTAAATTTAACTATCCAAAATGATGCTTATATACCAAAA
TATGATTCTAATGGAACAAGTGATATAGAACAACATGATGTTAATGAACTTAATGTATTT
TTCTATTTAGATGCACAGAAAGTGCCCGAAGGTGAAAATAATGTCAATCTCACCTCTTCA
ATTGATACAGCATTATTAGAACAACCTAAAATATATACATTTTTTTCATCAGAATTTATT
AATAATGTCAATAAACCTGTGCAAGCAGCATTATTTGTAAGCTGGATACAACAAGTGTTA
GTAGATTTTACTACTGAAGCTAACCAAAAAAGTACTGTTGATAAAATTGCAGATATTTCT
ATAGTTGTTCCATATATAGGTCTTGCTTTAAATATAGGAAATGAAGCACAAAAAGGAAAT
TTTAAAGATGCACTTGAATTATTAGGAGCAGGTATTTTATTAGAATTTGAACCCGAGCTT
TTAATTCCTACAATTTTAGTATTCACGATAAAATCTTTTTTAGGTTCATCTGATAATAAA
AATAAAGTTATTAAAGCAATAAATAATGCATTGAAAGAAAGAGATGAAAAATGGAAAGAA
GTATATAGTTTTATAGTATCGAATTGGATGACTAAAATTAATACACAATTTAATAAAAGA
AAAGAACAAATGTATCAAGCTTTACAAAATCAAGTAAATGCAATTAAAACAATAATAGAA
TCTAAGTATAATAGTTATACTTTAGAGGAAAAAAATGAGCTTACAAATAAATATGATATT
AAGCAAATAGAAAATGAACTTAATCAAAAGGTTTCTATAGCAATGAATAATATAGACAGG
TTCTTAACTGAAAGTTCTATATCCTATTTAATGAAATTAATAAATGAAGTAAAAATTAAT
AAATTAAGAGAATATGATGAGAATGTCAAAACGTATTTATTGAATTATATTATACAACAT
GGATCAATCTTGGGAGAGAGTCAGCAAGAACTAAATTCTATGGTAACTGATACCCTAAAT
AATAGTATTCCTTTTAAGCTTTCTTCTTATACAGATGATAAAATTTTAATTTCATATTTT
AATAAATTCTTTAAGAGAATTAAAAGTAGTTCAGTTTTAAATATGAGATATAAAAATGAT
AAATACGTAGATACTTCAGGATATGATTCAAATATAAATATTAATGGAGATGTATATAAA
TATCCAACTAATAAAAATCAATTTGGAATATATAATGATAAACTTAGTGAAGTTAATATA
TCTCAAAATGATTACATTATATATGATAATAAATATAAAAATTTTAGTATTAGTTTTTGG
GTAAGAATTCCTAACTATGATAATAAGATAGTAAATGTTAATAATGAATACACTATAATA
AATTGTATGAGAGATAATAATTCAGGATGGAAAGTATCTCTTAATCATAATGAAATAATT
TGGACATTGCAAGATAATGCAGGAATTAATCAAAAATTAGCATTTAACTATGGTAACGCA
AATGGTATTTCTGATTATATAAATAAGTGGATTTTTGTAACTATAACTAATGATAGATTA
GGAGATTCTAAACTTTATATTAATGGAAATTTAATAGATCAAAAATCAATTTTAAATTTA
GGTAATATTCATGTTAGTGACAATATATTATTTAAAATAGTTAATTGTAGTTATACAAGA
TATATTGGTATTAGATATTTTAATATTTTTGATAAAGAATTAGATGAAACAGAAATTCAA
ACTTTATATAGCAATGAACCTAATACAAATATTTTGAAGGATTTTTGGGGAAATTATTTG
CTTTATGACAAAGAATACTATTTATTAAATGTGTTAAAACCAAATAACTTTATTGATAGG
AGAAAAGATTCTACTTTAAGCATTAATAATATAAGAAGCACTATTCTTTTAGCTAATAGA
TTATATAGTGGAATAAAAGTTAAAATACAAAGAGTTAATAATAGTAGTACTAACGATAAT
CTTGTTAGAAAGAATGATCAGGTATATATTAATTTTGTAGCCAGCAAAACTCACTTATTT
CCATTATATGCTGATACAGCTACCACAAATAAAGAGAAAACAATAAAAATATCATCATCT
GGCAATAGATTTAATCAAGTAGTAGTTATGAATTCAGTAGGAAATAATTGTACAATGAAT
TTTAAAAATAATAATGGAAATAATATTGGGTTGTTAGGTTTCAAGGCAGATACTGTAGTT
GCTAGTACTTGGTATTATACACATATGAGAGATCATACAAACAGCAATGGATGTTTTTGG
AACTTTATTTCTGAAGAACATGGATGGCAAGAAAAATAA
Claims (22)
- 配列番号:1の核酸配列に対して少なくとも90%の配列同一性を有し且つ単鎖BoNT/E1タンパク質をコードする近接ヌクレオチドの配列を含む核酸配列。
- 前記核酸配列は、最大160個の低速コドンを有する、請求項1記載の核酸配列。
- 前記単鎖BoNT/E1タンパク質は、配列番号:2のアミノ酸配列に対して少なくとも95%の配列同一性を有する近接アミノ酸の配列を含む、請求項1又は2記載の核酸配列。
- 請求項1〜3の何れかに記載の核酸配列: ここで、請求項1又は2記載の単鎖BoNT/E1又は請求項3記載の近接アミノ酸の前記配列は、以下のアミノ酸のうち1個以上を含む(アミノ酸位置の番号付けは、前記BoNT/E1タンパク質のN末端アミノ酸残基から開始され、C末端アミノ酸残基で終了する):
位置177のグリシン、
位置198のセリン、
位置340のアラニン、
位置773のロイシン、
位置963のロイシン、
位置964のグルタミン、
位置967のアラニン、及び
位置1195のアスパラギン。 - 近接ヌクレオチドの前記配列は、野生型BoNT/E1(配列番号:3)の核酸配列と比較した時、少なくとも785個の同義コドンを有する、何れかの先行請求項記載の核酸配列。
- E. coli宿主細胞において可溶性単鎖BoNT/E1タンパク質を製造する方法であって、
E. coli発現系において、何れかの先行請求項記載の核酸配列を発現させることを含む方法。 - 前記可溶性単鎖BoNT/E1タンパク質は、前記E. coli宿主細胞の細胞質において発現する、請求項6記載の方法。
- 前記可溶性単鎖BoNT/E1タンパク質は、少なくとも5mg/Lのレベルで発現する、請求項6又は7記載の方法。
- 前記可溶性単鎖BoNT/E1タンパク質を含むE. coli宿主細胞ホモジネートを提供するE. coli宿主細胞の溶解を含む、請求項6〜8の何れかに記載の方法。
- 可溶性2本鎖BoNT/E1タンパク質を製造する方法であって、
配列番号:2のアミノ酸配列に対して少なくとも95%の配列同一性を有する近接アミノ酸の配列を含む可溶性単鎖BoNT/E1タンパク質を提供することと、前記BoNT/E1タンパク質を溶液中でトリプシンに接触させることとを含む、方法。 - 近接アミノ酸の前記配列は、以下のアミノ酸のうち1個以上を含む、請求項10記載の方法(アミノ酸位置の番号付けは、前記BoNT/E1タンパク質のN末端アミノ酸残基から開始され、C末端アミノ酸残基で終了する):
位置177のグリシン、
位置198のセリン、
位置340のアラニン、
位置773のロイシン、
位置963のロイシン、
位置964のグルタミン、
位置967のアラニン、及び
位置1195のアスパラギン。 - 前記可溶性単鎖BoNT/E1タンパク質は、請求項6〜9の何れかに記載の方法により提供される、請求項10又は11記載の方法。
- 可溶性BoNT/E1タンパク質及びトリプシンを含む溶液を、前記可溶性BoNT/E1タンパク質が優先的に結合する疎水性表面に接触させることにより、前記可溶性BoNT/E1タンパク質をトリプシンから分離することを含む、請求項10〜12の何れかに記載の方法。
- 前記疎水性表面は、アリール又はアルキル基からなるリガンドが付着した不活性マトリックスである、請求項13記載の方法。
- 前記疎水性表面は、ブチル、フェニル、又はオクチルリガンドからなる群から選択される、請求項14記載の方法。
- 活性2本鎖BoNT/E1タンパク質であって、
第1の鎖は、配列番号:2の位置1〜419のアミノ酸配列に対して少なくとも95%の配列同一性を有する近接アミノ酸の配列を含み、
第2の鎖は、配列番号:2の位置423〜1252のアミノ酸配列に対して少なくとも95%の配列同一性を有する近接アミノ酸の配列を含み、
前記第1及び第2の鎖は、前記第1の鎖上のシステイン412と前記第2の鎖上のシステイン426との間のジスルフィド結合により互いに連結され、
条件として、近接アミノ酸の前記配列は、以下のアミノ酸のうち1個以上を含む、活性2本鎖BoNT/E1タンパク質(アミノ酸位置の番号付けは、BoNT/E1タンパク質のN末端アミノ酸残基から開始され、C末端アミノ酸残基で終了する):
位置177のグリシン、
位置198のセリン、
位置340のアラニン、
位置773のロイシン、
位置963のロイシン、
位置964のグルタミン、
位置967のアラニン、及び
位置1195のアスパラギン。 - 請求項10〜15の何れかに記載の方法により得られる活性2本鎖BoNT/E1タンパク質。
- 請求項16又は17記載の活性2本鎖BoNT/E1タンパク質、又は請求項6〜9の何れかに記載の単鎖BoNT/E1タンパク質のタンパク質切断により得られる活性2本鎖BoNT/E1タンパク質を含み、トリプシンを実質的に含まない組成物。
- 前記組成物は、BoNT/E1タンパク質100ng当たり10pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり7pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり5pg未満のトリプシンを含む、請求項18記載の組成物。
- 液体医薬組成物であって、
請求項16又は17記載の活性2本鎖BoNT/E1タンパク質、又は請求項6〜9の何れかに記載の単鎖BoNT/E1タンパク質のタンパク質切断により得られる活性2本鎖BoNT/E1タンパク質と、
界面活性剤である非タンパク質安定化剤と、
水と、を含み、
タンパク質を安定化剤として含まず、
トリプシンを実質的に含まない(例えば、BoNT/E1タンパク質100ng当たり10pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり7pg未満のトリプシン、又はBoNT/E1タンパク質100ng当たり5pg未満のトリプシンを含む)、液体医薬組成物。 - 更に、
塩化ナトリウムと、
pHを5.5〜7.5に維持する緩衝剤と、
二糖と、を含み
前記水は滅菌水である、請求項20記載の液体医薬組成物。 - 治療において使用するための、請求項16又は17記載の活性2本鎖BoNT/E1タンパク質、又は請求項6〜9の何れかに記載の単鎖BoNT/E1タンパク質のタンパク質切断により得られる活性2本鎖BoNT/E1タンパク質、又は請求項18又は19記載の組成物、又は請求項20又は21記載の液体医薬組成物。
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