JP6384037B2 - 標的素材結合ペプチドおよびそのスクリーニング方法 - Google Patents
標的素材結合ペプチドおよびそのスクリーニング方法 Download PDFInfo
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- JP6384037B2 JP6384037B2 JP2013222610A JP2013222610A JP6384037B2 JP 6384037 B2 JP6384037 B2 JP 6384037B2 JP 2013222610 A JP2013222610 A JP 2013222610A JP 2013222610 A JP2013222610 A JP 2013222610A JP 6384037 B2 JP6384037 B2 JP 6384037B2
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- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
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- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
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- 125000004368 propenyl group Chemical group C(=CC)* 0.000 description 1
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- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
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- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- URLJMZWTXZTZRR-UHFFFAOYSA-N sodium myristyl sulfate Chemical compound CCCCCCCCCCCCCCOS(O)(=O)=O URLJMZWTXZTZRR-UHFFFAOYSA-N 0.000 description 1
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- CSMFSDCPJHNZRY-UHFFFAOYSA-N sulfuric acid monodecyl ester Natural products CCCCCCCCCCOS(O)(=O)=O CSMFSDCPJHNZRY-UHFFFAOYSA-N 0.000 description 1
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- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
現在までに、酸化チタン認識ペプチドと融合された金属内包性タンパク質フェリチンを利用して、シリコン基板上にナノ粒子を配置し、そのナノ粒子の上に酸化チタン膜または酸化シリコン膜を形成させ、その酸化膜の上にナノ粒子を配置することによる、酸化膜とナノ粒子とを積層する技術が知られている(特許文献6)。さらに、酸化チタンと融合された金属内包性タンパク質フェリチンを利用して、コバルトや酸化鉄の金属ナノ粒子を内包するタンパク質をチタンで描かれたパターンの上に配向させた例も報告されている(非特許文献12)。
〔1〕AXPQKXXXXXXS(配列番号1)のアミノ酸配列〔Xは各々独立してアミノ酸残基を表す〕を含み、かつ標的素材に対する結合活性を有するペプチド。
〔2〕Xが、各々独立して、式:−NH−C(R)(H)−CO−〔式中、Rは、水素原子または置換されていてもよいC1〜C6アルキル基を表す〕により表されるアミノ酸残基である、〔1〕のペプチド。
〔3〕Xが各々独立してグリシン残基またはアラニン残基を表す、〔1〕または〔2〕のペプチド。
〔4〕配列番号1のアミノ酸配列が、AYPQKFXXXFMS(配列番号2)のアミノ酸配列〔Xは各々独立してアミノ酸残基を表す〕である、〔1〕〜〔3〕のいずれかのペプチド。
〔5〕以下からなる群より選ばれるペプチド:
1)AYPQKFNNNFMS(配列番号3)のアミノ酸配列を含むペプチド;
2)AAPQKFNNNFMS(配列番号4)のアミノ酸配列を含むペプチド;
3)AYPQKANNNFMS(配列番号5)のアミノ酸配列を含むペプチド;
4)AYPQKFANNFMS(配列番号6)のアミノ酸配列を含むペプチド;
5)AYPQKFNGNFMS(配列番号7)のアミノ酸配列を含むペプチド;
6)AYPQKFNNAFMS(配列番号8)のアミノ酸配列を含むペプチド;
7)AYPQKFNNNAMS(配列番号9)のアミノ酸配列を含むペプチド;
8)AYPQKFNNNFAS(配列番号10)のアミノ酸配列を含むペプチド;
9)ATGSRMDHNRYI(配列番号11)のアミノ酸配列を含むペプチド;
10)DLLAMHWNTSRQ(配列番号12)のアミノ酸配列を含むペプチド;
11)IQAATVPHVTES(配列番号13)のアミノ酸配列を含むペプチド;
12)KVKHPSSWAYYA(配列番号14)のアミノ酸配列を含むペプチド;
13)SNSIDKVNRPIN(配列番号15)のアミノ酸配列を含むペプチド;
14)配列番号3〜15のアミノ酸配列のいずれか一つのアミノ酸配列において1個または2個のアミノ酸残基が欠失、置換、付加または挿入されたアミノ酸配列を含み、かつ、標的素材に対する結合活性を有する、ペプチド;ならびに
15)配列番号3〜15のアミノ酸配列のいずれか一つのアミノ酸配列に対して70%以上の同一性を示すアミノ酸配列を含み、かつ、標的素材に対する結合活性を有する、ペプチド。
〔6〕標的素材が無機素材である、〔1〕〜〔5〕のいずれかのペプチド。
〔7〕無機素材がチタン金属またはチタン化合物である、〔6〕のペプチド。
〔8〕〔1〕〜〔7〕のいずれかのペプチドとタンパク質との融合タンパク質。
〔9〕内腔を有する多量体を形成し得るポリペプチド部分、ならびに標的物質に結合し得るペプチド部分および〔1〕〜〔7〕のいずれかのペプチドの部分を含む、融合タンパク質。
〔10〕内腔を有する多量体を形成し得るポリペプチド部分がDpsである、〔9〕の融合タンパク質。
〔11〕標的物質が、金属素材、シリコン素材または炭素素材である、〔9〕または〔10〕の融合タンパク質。
〔12〕炭素素材がカーボンナノ素材である、〔11〕の融合タンパク質。
〔13〕融合タンパク質の多量体であって、
内腔を有し、
融合タンパク質が、内腔を有する多量体を形成し得るポリペプチド部分、ならびに標的物質に結合し得るペプチド部分および〔1〕〜〔7〕のいずれかのペプチドの部分を含む、多量体。
〔14〕複合体であって、
〔13〕の多量体、ならびに標的物質およびチタンを含み、
標的物質が、前記融合タンパク質中の標的物質に結合し得るペプチド部分に結合し、かつチタンが、前記融合タンパク質中のチタン結合ペプチドの部分に結合している、複合体。
〔15〕〔8〕〜〔12〕のいずれかの融合タンパク質をコードするポリヌクレオチド。
〔16〕〔15〕のポリヌクレオチドを含む発現ベクター。
〔17〕〔16〕の発現ベクターを含む形質転換体。
〔18〕形質転換体がエシェリヒア・コリである、〔17〕の形質転換体。
〔19〕以下を含む、標的素材に結合し得るペプチドのスクリーニング方法:
1)標的素材をファージライブラリに接触させて、ファージ結合標的素材を得ること;
2)ファージ結合標的素材を回収すること;
3)ファージ結合標的素材および宿主を用いて、ファージ導入宿主を調製すること;
4)ファージ導入宿主において、ファージを増幅させること;および
5)増幅したファージが発現する、標的素材に結合し得るペプチドを同定すること。
〔20〕回収したファージ結合標的素材の洗浄後に、ファージ結合標的素材を宿主に導入する、〔19〕の方法。
〔21〕前記1)〜4)のサイクルを複数回繰り返した後に、前記5)の工程を行う、〔19〕または〔20〕の方法。
〔22〕標的素材がナノ素材である、〔19〕〜〔21〕のいずれかの方法。
本発明の融合タンパク質、多量体または複合体は、例えば、電気特性および/または光触媒活性の向上した新規デバイスの作製、ならびに医療、バイオ研究分野等の分野に有用である。例えば、酸化チタンに結合し得るペプチド部分およびカーボンナノチューブに結合し得るペプチド部分を含む融合タンパク質の使用により、抗菌素材、脱臭素材、大気浄化素材、防汚性素材、水素発生装置、太陽電池、半導体などの提供が可能になる。
本発明のスクリーニング方法は、例えば、標的素材に強固に結合する能力を有するペプチドの効率的な同定に有用である。
1)AYPQKFNNNFMS(配列番号3)のアミノ酸配列を含むペプチド;
2)AAPQKFNNNFMS(配列番号4)のアミノ酸配列を含むペプチド;
3)AYPQKANNNFMS(配列番号5)のアミノ酸配列を含むペプチド;
4)AYPQKFANNFMS(配列番号6)のアミノ酸配列を含むペプチド;
5)AYPQKFNGNFMS(配列番号7)のアミノ酸配列を含むペプチド;
6)AYPQKFNNAFMS(配列番号8)のアミノ酸配列を含むペプチド;
7)AYPQKFNNNAMS(配列番号9)のアミノ酸配列を含むペプチド;
8)AYPQKFNNNFAS(配列番号10)のアミノ酸配列を含むペプチド;
9)ATGSRMDHNRYI(配列番号11)のアミノ酸配列を含むペプチド;
10)DLLAMHWNTSRQ(配列番号12)のアミノ酸配列を含むペプチド;
11)IQAATVPHVTES(配列番号13)のアミノ酸配列を含むペプチド;
12)KVKHPSSWAYYA(配列番号14)のアミノ酸配列を含むペプチド;
13)SNSIDKVNRPIN(配列番号15)のアミノ酸配列を含むペプチド;
14)配列番号3〜15のアミノ酸配列のいずれか一つのアミノ酸配列において1個または2個のアミノ酸残基が欠失、置換、付加または挿入されたアミノ酸配列を含み、かつ、チタン結合活性を有する、ペプチド;ならびに
15)配列番号3〜15のアミノ酸配列のいずれか一つのアミノ酸配列に対して70%以上の同一性を示すアミノ酸配列を含み、かつ、チタン結合活性を有する、ペプチド。
チタン結合活性は、上述したとおりである。
本発明のスクリーニング方法は、以下を含む:
1)標的素材をファージライブラリに接触させて、ファージ結合標的素材を得ること;
2)ファージ結合標的素材を回収すること;
3)ファージ結合標的素材および宿主を用いて、ファージ導入宿主を調製すること;
4)ファージ導入宿主において、ファージを増幅させること;および
5)増幅したファージが発現する、標的素材に結合し得るペプチドを同定すること。
(ファージパニング工程)
酸化チタン吸着ペプチドのスクリーニングは、Ph.D.TM−12 Phage Display Libraries(NEW ENGLAND BioLabs Inc.、米国)を用いた。すなわち、酸化チタンナノ粒子(Titanium(IV) oxide,anatase、直径25nm、Sigma Aldrich)を終濃度0.01mg/ml(4x1010個/ml)で含有するTBT01緩衝液(50mM TrisHCl,pH8.0,0.1容量% tween−20含有)1mlに、最終的なファージ濃度が1011個/mlとなるようにPh.D.TM−12 Phage Display Librariesのファージ溶液を添加し、室温で遮光しながら1時間回転放置した。反応後、酸化チタンナノ粒子を遠心回収し、TBT02緩衝液(50mM TrisHCl,pH8.0,0.2容量% tween−20含有)1mlに再度懸濁した。この酸化チタンナノ粒子を遠心回収し、新たな緩衝液1mlに懸濁する工程を、tween−20を0.3容量%、0.4容量%、そして0.6容量%の濃度でそれぞれ含むトリス緩衝液(50mM TrisHCl,pH8.0)を用いて実施した。最後に水で酸化チタンナノ粒子を2回洗浄し、水110μlに懸濁した。これらの工程により、サンプルに含まれる酸化チタンに結合していないファージが多くとも100個以下になるようにした。
今回のスクリーニングで得られたST1(AYPQKFNNNFMS(配列番号3))と従来知られていたチタン結合ペプチドminTBP1(RKLPDA(配列番号49)、国際公開第2005/010031号、Sano et al.(2005) Langmuir.21,p.3090)を化学合成(SigmaAldrich社カスタムペプチド合成サービス使用)した。そして、チタンセンサーを使った水晶マイクロバランス振動法(QCM)を用いて、各ペプチドのチタンへの結合能力を評価した。
表3に示すチタン結合ペプチドがC末端に融合されたListeria innocuaの金属内包性タンパク質Dpsを下記の手順によりそれぞれ構築した。
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号50)
5’−tttGGATCCttaCGACATAAAATTATTATTAAACTTCTGAGGATAAGCttctaatggagcttttc−3’(配列番号51)
Dps−Ti1をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号52)
5’−tttGGATCCTTAccagccaccgcccagtttatgggtcgggtttttggtgcgctttttacgttctaatggagcttttc−3’ (配列番号53)
Dps−Ti2をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号54)
5’−tttGGATCCTTAccagccaccgcctttcaggctgctcggaaagcggcgaatcatgcgcatttctaatggagcttttc−3’(配列番号55)
Dps−Ti3をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号56)
5’−tttGGATCCTTAccagccAccgccatgGtgatgaatGtgatcatggcggctcaggcttttttctaatggagcttttc−3’(配列番号57)
Dps−Ti4をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号58)
5’−tttGGATCCTTAccagccAccgcctttggtcgcatagcgcggatggctcagatgctgggtttctaatggagcttttc−3’(配列番号59)
Dps−Ti5をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号60)
5’−tttGGATCCttaccagccgccgccggtcgggctcgcgctcggctggcggctcgggcggctttctaatggagcttttc−3’(配列番号61)
Dps−Ti731をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号62)
5’−tttGGATCCTTAatacgggctcacccaggtcgcttctaatggagcttttc−3’(配列番号63)
Dps−Ti(Si)をコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号64)
5’−tttGGATCCttaGGTGTTGGTCAGCCACTGTGGCGGCGCGCTCGGATAttctaatggagcttttc−3’(配列番号65)
Dps−Vreulsをコードする遺伝子が搭載されたベクター
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号66)
5’−tttGGATCCTTAgctgcccgccatggtaaacggcacAGCcgcgttcagttctaatggagcttttc−3’(配列番号67)
5’−tttGGATCCGAATTCGAGCTCCGTCG−3’(配列番号68)
5’−tttGGATCCttaCGACATAAAATTATTATTAAACTTCTGAGGATAAGCttctaatggagcttttc−3’(配列番号69)
発現プラスミド(pET20−Dps−ST1、pET20−Dps−Ti2、pET20−Dps−Ti3、pET20−Dps−Ti4、pET20−Dps−Ti5、pET20−Dps−Ti731、pET20−Dps−Ti(Si)、pET20−Dps−Vreuls、pET20−CDTS1)のいずれかを保持したBL21(DE3)を50mLのLB培地(100mg/L アンピシリンを含む)にて37℃で振とう培養した。培養開始24時間後、得られた菌体を遠心分離(6000rpm、5分間)により回収し、50mM TrisHCl緩衝液(pH8.0) 10mLで懸濁した。次に、その懸濁液にDigital Sonifier 450(Branson社、USA)を使い超音波パルス(200W、Duty 45%)を5分間与えることで、菌体を破砕した。その溶液を6000rpmで5分間、遠心分離し、上清画分を回収した。回収された溶液を60℃で20分間加熱し、加熱後は速やかに氷上で冷却した。冷却された溶液を6000rpmで5分間、遠心分離し、再度、上清を回収(約10mL)した。その溶液をディスクフィルター(Millex GP 0.22μm、Millipore社、USA)で粗い粒子を除去し、タンパク質粗抽出液を得た。
続いて、各ペプチドのチタン析出能力を評価するために、各ペプチドと融合したDpsのチタン析出能力を評価した。すなわち、変異型Dps(Dps−ST1、Dps−Ti2、Dps−Ti3、Dps−Ti4、Dps−Ti5、Dps−Ti731、Dps−Ti(Si)、Dps−Vreuls、CDT1)を各々終濃度0.01mg/ml、Titanium(IV) bis(ammonium lactato)dihydroxide(SigmaAldrich社、388165)を終濃度が3.0重量%となるように加えた50mMリン酸緩衝液(pH6.0)に加え、室温で6時間放置した後、660nmの吸光を測定し、タンパク質のみを含まない溶液をバックグラウンドとした。反応容量は0.2mlとして実験を行った。
その結果、今回測定されたすべての変異型Dps(Dps−ST1、CDT1、Dps−Ti5)において時間と共に吸光度が上昇する様子を確認できた(図3)。そして、Dps−ST1は、他のペプチドを提示したDpsと比較して、約2倍チタン析出活性が高いことが示唆された(図4)。
変異型Dps(Dps−ST1、Dps−minTBP1、Dps−Ti5、Dps、CDTS1、CDT1、CD1)を用いてST1のチタン結合能力を、チタンセンサーを使った水晶マイクロバランス振動法(QCM)により評価した。
CNTとCDTS1とのナノ複合体を形成するために、CNTとCDTS1とを含むリン酸カリウム緩衝液[50mM リン酸カリウム(pH6.0)、0.2mg/mLのCDTS1、及び0.1mg/mL CNT(Sigma社、519308, carbon nanotube, single walled)を各々終濃度で含む]を調製した。CDTS1を含むリン酸カリウム緩衝液に、Digital Sonifier 450(Branson社、USA)を用いて、氷上で、3秒間隔で1秒間の超音波パルス(200W、Duty 20%)を合計5分間加えた。超音波処理されたCDTS1−CNT混合溶液を遠心分離(15000rpm、5分間)し、沈殿を回収し、多数のCDTS1がCNTに結合したCNT/CDTS1複合体を得た。
得られたCNT/CDTS1複合体溶液に、終濃度2.5wt%となるようにチタン前駆体Titanium(IV) bis(ammonium lactato)dihydroxide(SIGMA社、388165)を加え、室温(24℃)で放置した。そして、反応を開始して6時間後のサンプルを遠心分離(15000rpm、5分間)して、沈殿を回収した。得られた沈殿を水で3回洗浄し、最後に水に懸濁した。得られたサンプルを、3%リンタングステン酸で染色し、透過型電子顕微鏡(JEM3100−FEF、300kV)にて、電子顕微鏡解析した。
よって、チタン前駆体が添加されたCNT/CDTS1ナノ複合体溶液を観察したときに見られるロッド状の構造物には少なくとも、炭素原子とチタン原子が含まれていることが分かった。この結果は、CNTと結合したCDTS1のチタン析出活性を利用することで、CNT/CDTS1の複合体(ナノ複合体)の周囲をチタンで被膜し、CNT/CDTS1/Tiの複合体(ナノ複合体)が合成できることがわかった。
ST1(AYPQKFNNNFMS(配列番号3))のチタン結合能力に必要なアミノ酸を特定すべく、ST1ペプチド配列内のアミノ酸をそれぞれアラニン(A)あるいはグリシン(G)に置き換えられた配列を持つペプチドがC末端に融合されたListeria innocuaの金属内包性タンパク質Dpsを下記の手順によりそれぞれ構築した。各変異DpsがC末端に持つペプチド配列と構築に用いた特異的プライマーを表4に示す。
得られたPCR産物をWizard SV Gel and PCR Clean−Up System(Promega社、USA)で精製し、制限酵素DpnIとBamHIで消化した。制限酵素で消化されたPCR産物を、T4 DNAリガーゼ(Promega社、USA)を用いてセルフライゲーションさせた。セルフライゲーションされたPCR産物をECOSTM Competent E.coli BL21(DE3)(ニッポンジーン社、日本)に形質転換し、C末端に各チタン結合ペプチドが融合されたDpsをコードする遺伝子が搭載された発現プラスミド(pET20−Dps−ST1−1G、pET20−Dps−ST1−2A、pET20−Dps−ST1−3A、pET20−Dps−ST1−4A、pET20−Dps−ST1−5A、pET20−Dps−ST1−6A、pET20−Dps−ST1−7A、pET20−Dps−ST1−8G、pET20−Dps−ST1−9A、pET20−Dps−ST1−10A、pET20−Dps−ST1−11A、pET20−Dps−ST1−12A)を保持したBL21(DE3)を構築した。その形質転換株からWizard Plus Minipreps System(Promega社、USA)を使って各ベクターを精製し、搭載された変異Dps遺伝子配列を確認した。SDS−PAGE解析により、Dps−ST1−1G、Dps−ST1−2A、Dps−ST1−3A、Dps−ST1−4A、Dps−ST1−5A、Dps−ST1−6A、Dps−ST1−7A、Dps−ST1−8G、Dps−ST1−9A、Dps−ST1−10A、Dps−ST1−11A、Dps−ST1−12Asの発現を確認でき、タンパク質発現用株を得ることができた。得られたタンパク質はDps−ST1やCDTS1と同様にして精製し、濃縮後、濃度決定された。
先に構築された変異型Dps−ST1(ST1:AYPQKFNNNFMS(配列番号3))ペプチド配列内のアミノ酸がそれぞれ1つずつ、アラニン(A)あるいはグリシン(G)に置き換えられた配列を持つペプチドがC末端に融合されたDps)のチタンへの結合能力を、チタンセンサーを使った水晶マイクロバランス振動法(QCM)により評価した。
Claims (16)
- 以下からなる群より選ばれるペプチド:
1)AYPQKFNNNFMS(配列番号3)のアミノ酸配列を含むペプチド;
2)AAPQKFNNNFMS(配列番号4)のアミノ酸配列を含むペプチド;
3)AYPQKANNNFMS(配列番号5)のアミノ酸配列を含むペプチド;
4)AYPQKFANNFMS(配列番号6)のアミノ酸配列を含むペプチド;
5)AYPQKFNGNFMS(配列番号7)のアミノ酸配列を含むペプチド;
6)AYPQKFNNAFMS(配列番号8)のアミノ酸配列を含むペプチド;
7)AYPQKFNNNAMS(配列番号9)のアミノ酸配列を含むペプチド;
8)AYPQKFNNNFAS(配列番号10)のアミノ酸配列を含むペプチド;
9)ATGSRMDHNRYI(配列番号11)のアミノ酸配列を含むペプチド;
10)DLLAMHWNTSRQ(配列番号12)のアミノ酸配列を含むペプチド;
11)IQAATVPHVTES(配列番号13)のアミノ酸配列を含むペプチド;
12)KVKHPSSWAYYA(配列番号14)のアミノ酸配列を含むペプチド;および
13)SNSIDKVNRPIN(配列番号15)のアミノ酸配列を含むペプチド。 - 請求項1記載のペプチドとタンパク質との融合タンパク質。
- 内腔を有する多量体を形成し得るポリペプチド部分と、標的物質に結合し得るペプチド部分と、請求項1記載のペプチドの部分とを含む、融合タンパク質。
- 内腔を有する多量体を形成し得るポリペプチド部分がDpsである、請求項3記載の融合タンパク質。
- 標的物質が、金属素材、シリコン素材または炭素素材である、請求項3または4記載の融合タンパク質。
- 炭素素材がカーボンナノ素材である、請求項5記載の融合タンパク質。
- 融合タンパク質の多量体であって、
内腔を有し、
融合タンパク質が、内腔を有する多量体を形成し得るポリペプチド部分と、標的物質に結合し得るペプチド部分と、請求項1記載のペプチドの部分とを含む、多量体。 - 複合体であって、
請求項7記載の多量体と、標的物質と、チタンとを含み、
標的物質が、前記融合タンパク質中の標的物質に結合し得るペプチド部分に結合し、かつチタンが、前記融合タンパク質中のチタン金属またはチタン化合物に対する結合活性を有するペプチドの部分に結合している、複合体。 - 請求項2〜6のいずれか一項記載の融合タンパク質をコードするポリヌクレオチド。
- 請求項9記載のポリヌクレオチドを含む発現ベクター。
- 請求項10記載の発現ベクターを含む形質転換体。
- 形質転換体がエシェリヒア・コリである、請求項11記載の形質転換体。
- 以下を含む、標的素材に結合し得るペプチドのスクリーニング方法:
1)標的素材をファージライブラリに接触させて、ファージ結合標的素材を得ること;
2)ファージ結合標的素材を回収すること;
3)ファージ結合標的素材および宿主を用いて、エレクトロポレーション法により、ファージ導入宿主を調製すること;
4)ファージ導入宿主において、ファージを増幅させること;および
5)増幅したファージが発現する、標的素材に結合し得るペプチドを同定すること。 - 回収したファージ結合標的素材の洗浄後に、ファージ導入宿主を調製する、請求項13記載の方法。
- 前記1)〜4)のサイクルを複数回繰り返した後に、前記5)の工程を行う、請求項13または14記載の方法。
- 標的素材がナノ素材である、請求項13〜15のいずれか一項記載の方法。
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