JP4907542B2 - 治療、診断およびクロマトグラフィーに使用するためのタンパク質複合体 - Google Patents
治療、診断およびクロマトグラフィーに使用するためのタンパク質複合体 Download PDFInfo
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- JP4907542B2 JP4907542B2 JP2007535119A JP2007535119A JP4907542B2 JP 4907542 B2 JP4907542 B2 JP 4907542B2 JP 2007535119 A JP2007535119 A JP 2007535119A JP 2007535119 A JP2007535119 A JP 2007535119A JP 4907542 B2 JP4907542 B2 JP 4907542B2
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- 230000003595 spectral effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000011191 terminal modification Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
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- 101150062838 ubl-1 gene Proteins 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
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- 230000009385 viral infection Effects 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
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Description
1.ウシタンパク質と比較して、ヒトγ−II−クリスタリンは、変性作用に対して著しく高い安定性を有している。
2.ヒト由来タンパク質は、治療的応用において、各変異の免疫原生が非常に低いという結果をもたらす。そして、
3.より高度な複雑性を有する新しく構築されたライブラリは、さらに高い親和性を有する結合分子の単離を可能とするであろう(非特許文献13)。
前記GCUC1ライブラリと同様に、同じ8個のアミノ酸部位(開始メチオニンを除く、2、4、6、15、17、19、36および38部位)が、ランダム化のために選択された。新しいライブラリCR20は、それぞれ独立した理論上5×108通りの変異体を有するヒトγ−II−クリスタリンに基づき、特許文献1に記載された方法で確立され、前記ライブラリにおいて変異体は約130倍で表される。200以上の独立した変異体をシークエンスした後、全変異体の80%以上が、前記8箇所のランダム化部位においてのみ置換を有することがわかった。さらに、3番目のコドン部位を除く置換部位における変異の配列は、可能性のある全てのヌクレオチドと、ほとんど同一の分布を示した。このように、前記8箇所のランダム化部位における全32の可能性のあるコドンを有するこのライブラリは、質が高い。
ユビキチンまたはγ−クリスタリンに基づく1以上のポリペプチド(I)であって、対応する野生型ポリペプチドと比較して新たに発生または変化させた、リガンドに特異的に結合するための結合特性をそれぞれ有するポリペプチドと、
それに共有結合的に結合される1以上の機能性成分(II)とを含み、
前記(II)に対する前記(I)のカップリング後、全ての成分の機能性が維持されている、複合体。
a)修飾されたタンパク質の選択
b)結合パートナーの決定
c)βシート領域の少なくとも1つのβシート鎖および任意に非βシート領域を含むタンパク質の少なくとも1つの表面露出領域におけるアミノ酸の選択
d)ユビキチン様折りたたみモチーフが保持される間、置換、挿入、欠損および/または化学的な修飾により、選択されたアミノ酸の修飾
e)工程b)において決定された結合パートナーへの前記修飾タンパク質の接触
f)工程b)において決定された結合パートナーへの結合親和性を有するタンパク質の検出
タンパク質の空間構造の分析による、カップリングに適したアミノ酸残基、好ましくは、リガンドとの(I)の相互作用の表面の外側のアミノ酸残基の同定;
適したカップリング試薬によるカップリングパートナーの活性化;
カップリング反応の実行;
複合体の単離;および
複合体の両成分の機能性の検出
置換、挿入または融合によるカップリングに適したアミノ酸残基、好ましくは、リガンドとの(I)の相互作用の表面の外側表面に曝されるアミノ酸残基の導入;
導入されたアミノ酸残基の露出度の検出;
この方法により変化させた成分(I)の機能性の検出;
適したカップリング試薬によるカップリングパートナーの活性化;
カップリング反応の実行;
複合体の単離;および
複合体の両成分の機能性の検出
1.)デキストランマトリクスに対するC末端システインを介したAffilin(商標)SPU3−A1_Cysのカップリング、
2.)BIACOREシステムの前記デキストランマトリクスに対する第1級アミノ基を介したAffilin(商標)SPC7−E9のカップリング、および、
3.)EDC/NHSによるポリメタクリル酸マトリクスに対するAffilin(商標)SPC7−E9の非特異的なカップリング。
CR20 (SEQ ID NO:1)
ATGGGTNNKATCNNKTTCNNKGAAGACCGTGCTTTCCAGGGTCGTNNKTACNNKTGCNNKACCGACTGCCCGAACCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCNNKGTTNNKTCCGGTTGCTGGATGATCTACGAACGTCCGAACTACCAGGGTCACCgtcaccagtacttcctgcggcgtggggagtaccccgactaccagcaatggatgggcctcagcgactccatccgctcctgctgcctcatccccccccactctggcgcttacagaatgaagatctacgacagagatgaattgaggggacaaatgtcagagctcacagacgactgtctctctgttcaggaccgcttccacctcactgaaatt
cactccctcaatgtgctggagggcagctggatcctctatgagatgcccaactacagggggaggcagtatctgctgaggccgggggagtacaggaggtttcttgattggggggctccaaatgccaaagttggctctcttagacgagtcatggatttgtacgcg
SPC1-A1 (SEQ ID NO:2)
ATGGGTTTTATCTGGTTCATGGAAGACCGTGCTTTCCAGGGTCGTAGGTACGATTGCGGTACCGACTGCCCGAACCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCAAGGTTAAGTCCGGTTGCTGGATGATCTACGAACGTCCGAACTACCAGGGTCACCgtcaccagtacttcctgcggcgtggggagtaccccgactaccagcaatggatgggcctcagcgactccatccgctcctgctgcctcatccccccccactctggcgcttacagaatgaagatctacgacagagatgaattgaggggacaaatgtcagagctcacagacgactgtctctctgttcaggaccgcttccacctcactgaaattcactccctcaatgtgctggagggcagctggatcctctatgagatgcccaactacagggggaggcagtatctgctgaggccgggggagtacaggaggtttcttgattggggggctccaaatgccaaagttggctctcttagacgagtcatggatttgtacgcg
SPC1-A7 (SEQ ID NO:3)
ATGGGTCTGATCTGTTTCTCTGAAGACCGTGCTTTCCAGGGTCGTAGGTACATGTGCCTGACCGACTGCCCGAACCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCAATGTTTGGTCCGGTTGCTGGATGATCTACGAACGTCCGAACTACCAGGGTCACCgtcaccagtacttcctgcggcgtggggagtaccccgactaccagcaatggatgggcctcagcgactccatccgctcctgctgcctcatccccccccactctggcgcttacagaatgaagatctacgacagagatgaattgaggggacaaatgtcagagctcacagacgactgtctctctgttcaggaccgcttccacctcactgaaattcactccctcaatgtgctggagggcagctggatcctctatgagatgcccaactacagggggaggcagtatctgctgaggccgggggagtacaggaggtttcttgattggggggctccaaatgccaaagttggctctcttagacgagtcatggatttgtacgcg
SPC1-G3 (SEQ ID NO:4)
ATGGGTTCTATCATTTTCCTTGAAGACCGTGCTTTCCAGGGTCGTATTTACGGTTGCACTACCGACTGCCCGAACCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCGTGGTTCAGTCCGGTTGCTGGATGATCTACGAACGTCCGAACTACCAGGGTCACCgtcaccagtacttcctgcggcgtggggagtaccccgactaccagcaatggatgggcctcagcgactccatccgctcctgctgcctcatccccccccactctggcgcttacagaatgaagatctacgacagagatgaattgaggggacaaatgtcagagctcacagacgactgtctctctgttcaggaccgcttccacctcactgaaattcactccctcaatgtgctggagggcagctggatcctctatgagatgcccaactacagggggaggcagtatctgctgaggccgggggagtacaggaggtttcttgattggggggctccaaatgccaaagttggctctcttagacgagtcatggatttgtacgcg
SPC1-A7BB (SEQ ID NO:5)
ATGGGTCTGATCTCTTTCTCTGAAGACCGTGCTTTCCAGGGTCGTAGGTACATGTgCCTGACCGACTGCCCGAaCCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCAATGTTTGGTCCGGTTGCTGGATGATCTACGaACGTCCGAACTACCAGGGTCACCAGTACTTcCTGCGGCGTGGGGAGTACCCCGACTACCAGCAATGGATGGGCCTCAGCGACTCCATCCGCTCCTGCTGCCTCATCCCCCCCCACTCTGGCGCTTACAGAATGAAGATCTACGACAGAGATGAATTGAGGGGACAAATGTCAGAGCTCACAGACGACTGTCTCTCTGTTCAGGACCGCTTCCACCTCACTGAAATTCACTCCCTCAATGTGCTGGAGGGCAGCTGGATCCTCTATGAGATGCCCAACTACAGGGGGAGGCAGTATCTGCTGAGGCCGGGGGAGTACAGGAGGTTTCTTGATTGGGGGGCTCCAAATGCCAAAGTTGGCTCTCTTAGACGAGTCATGGATTTgTACCTCGAGCACCACCACCACCACCAC
SPC1-A7JJ (His10を含む) (SEQ ID NO:6)
ATGGGTCTGATCTCTTTCTCTGAAGACCGTGCTTTCCAGGGTCGTAGGTACATGTgCCTGACCGACTGCCCGAaCCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCAATGTTTGGTCCGGTTGCTGGATGATCTACGaACGTCCGAACTACCAGGGTCACCAGTACTTcCTGCGGCGTGGGGAGTACCCCGACTACCAGCAATGGATGGGCCTCAGCGACTCCATCCGCTCCTGCTGCCTCATCCCCCCCCACTCTGGCGCTTACAGAATGAAGATCTACGACAGAGATGAATTGAGGGGACAAATGTCAGAGCTCACAGACGACTGTCTCTCTGTTCAGGACCGCTTCCACCTCACTGAAATTCACTCCCTCAATGTGCTGGAGGGCAGCTGGATCCTCTATGAGATGCCCAACTACAGGGGGAGGCAGTATCTGCTGAGGCCGGGGGAGTACAGGAGGTTTCTTGATTGGGGGGCTCCAAATGCCAAAGTTGGCTCTCTTAGACGAGTCATGGATTTgTACCTCGAGTgCGGCGGCCATCACCATCACCACCACCACCACCACCAC
SPC1-A7_Cys (His10を含む) (SEQ ID NO:7)
ATGGGtCTGATCTCTtTcTcTGAAGACCGTGCTTTCCAGGGTCGTAGGtAcAtgTGCCTgAcCGacTgCccGAACCTGCAGCCGTACTTcTcCCGTTGCAACTCCATCAATGTTTGGTCCGGTTGCTGGATGATCTACGAACGTCCGAACTACCAGGGTCACCAGTACTTcCTGCGGCGTGGGGAGTACCCCGAcTACCAGCAATGGATGGGCCTCAGCGACTCCATCCGCTCCtGCTGCCTCATCCCCCCCCACTCTGGCGCTTACAGAATGAAGATCTACGACAGAGATGAATTGAGGGGACAAATGTCAGAGCTCACAGACGACTGTCTCTCTGTTCAGGACCGCTTCCACCTCACTGAAATTCACTCCCTCAATGTGCTGGAGGGCAGCTGGATCCTCTATGAGATGCCCAACTACAGGGGGAGGCAGTATCTGCTGAGGCCGGGGGAGTACAGGAGGTTTCTTGATTGGGGGGCTCCAAATGCCAAAGTTGGCTCTCTTAGACGaGTCATgGATTTgTACCTCGAGTCCGGCGGCGGGGGGGGAGGaTCTTgCCATCACCATCACCACCaCCACCAcCACCAC
SPC7-E9 (His6を含む) (SEQ ID NO:8)
ATGGGTTTTATCTGTTTCTTGGAAGACCGTGCTTTCCAGGGTCGTTCTTACGCTTGCGATACTGACTGCCCGAACCTGCAGCCGTACTTCTCCCGTTGCAACTCCATCAGTGTTCTGTCCGGTTGCTGGATGATCTACGAACGTCCGAACTACCAGGGTCACCAGTACTTCCTGCGGCGTGGGGAGTACCCCGACTACCAGCAATGGATGGGCCTCAGCGACTCCATCCGCTCCTGCTGCCTCATCCCCCCCCACTCTGGCGCTTACAGAATGAAGATCTACGACAGAGATGAATTGAGGGGACAAATGTCAGAGCTCACAGACGACTGTCTCTCTGTTCAGGACCGCTTCCACCTCACTGAAATTCACTCCCTCAATGTGCTGGAGGGCAGCTGGATCCTCTATGAGATGCCCAACTACAGGGGGAGGCAGTATCTgCTGAGGCCGGGGGAGTACAGGAGGTTTCTTGATTGGGGGGCTCCAAATGCCAAAGTTGGCTCTCTTAGACGAGTCATGGATTTGTACCTCGAGCACCACCACCACCACCAC
Ubiquitin wildtype (SEQ ID NO:9)
ATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCATCACTCTGGAGGTGGAGCCCAGTGACACCATCGAAAATGTGAAGGCCAAGATCCAAGATAAAGAAGGCATTCCCCCCGACCAGCAGAGGCTCATCTTTGCAGGCAAGCAGCTGGAAGATGGCCGCACTCTTTCTGACTACAACATCCAGAAAGAGTCGACCCTGCACCTGGTCCTCCGCCTGAGGGGCGGC
Modified ubiquitin (MUBI) (SEQ ID NO:10)
ATGCAAATCTTCGTTAAAACCCTGACGGGAAAGACTATCACCCTGGAGGTAGAACCGTCCGACACCATCGAAAATGTCAAAGCTAAAATCCAAGACAAAGAAGGAATTCCACCTGACCAGCAACGCCTAGCTTTCGCAGGACGACAACTAGAGGACGGGCTCACCCTGTCTGACTACAACATCCAAAAAGAATCCACCCTCCACCTGGCACTCCTCCTGCGGGCC
UB10 (library) (SEQ ID NO:11)
ATGNNKATCNNKGTTNNKACCCTGACGGGAAAGACTATCACCCTGGAGGTAGAACCGTCCGACACCATCGAAAATGTCAAAGCTAAAATCCAAGACAAAGAAGGAATTCCACCTGACCAGCAACGCCTAGCTTTCGCAGGACGACAACTAGAGGACGGGCTCACCCTGTCTGACTACAACATCNNKNNKNNKNNKNNKCTCCACCTGGCACTCCTCCTGCGGGCC
SPU11-3-A1 (His6を含む) (SEQ ID NO:12)
ATGCGGATCCGTGTTGCTACCCTGACGGGAAAGACTATCACCCTGGaGGTAGAACCGTCCGACACCATCGAAAATGTCAAAGCTAAAATCCAAGACAAAGAAGGAATTCCACCTGACCAGCAACGCCTAGCTTTCGCAGGACGACAACTAGAGGACGGGCTCACCCTGTCTGACTACGACATCCGTCATGGTACGTCGCTCCACCTGGcACTCCTCCTGCGGGCCCTCGAGCACCACCACCACCACCAC
SPU11-3-A1_Cys (His10を含む) (SEQ ID NO:13)
ATGCGGATCCGTGTTGCTACCCTGACGGGAAAGACTATCACCCTGGaGGTAGAACCGTCCGACACCATCGAAAATGTCAAAGCTAAAATCCAAGACAAAGAAGGAATTCCACCTGACCAGCAACGCCTAGCTTTCGCAGGACGACAACTAGAGGACGGGCTCACCCTGTCTGACTACGACATCCGTCATGGTACGTCGCTCCACCTGGcACTCCTCCTGCGGGCCCTCGAGTCCGGCGGCGGGGGGGGAGGATCTTGCCATCACCATCACCACCACCACCACCACCAC
pCAN700 (SEQ ID NO:14)
(5’-CCA TGA TTA CGC CAA GCT TTG GAG CC-3’)
A7Cys4Ser_for (SEQ ID NO:15)
(5’-CCA TGG GTC TGA TCT CTT TCT CTG AAG ACC G-3’)
A7Cys4Ser_rev (SEQ ID NO:16)
(5’-CGG TCT TCA GAG AAA GAG ATC AGA CCC ATG G-3’)
pETTerm (SEQ ID NO:17)
(5’-GCT AGT TAT TGC TCA GCG GTG GC-3’)
A7Gly2Cys_for (SEQ ID NO:18)
(5’-GGA TTT GTA CCT CGA GTG CGG CGG CCA TCA CCA TCA CCA CCA CCA CCA CCA CCA CTG AGA TCC GGC-3’)
A7Gly2Cys_rev (SEQ ID NO:19)
(5’-GCC GGA TCT CAG TGG TGG TGG TGG TGG TGG TGA TGG TGA TGG CCG CCG CAC TCG AGG TAC AAA TCC-3’)
A7Gly2Ser_for (SEQ ID NO:20)
(5’-GGA TTT GTA CCT CGA GTC CGG CGG CCA TCA CC-3’)
A7Gly2Ser rev (SEQ ID NO:21)
(5’-GGT GAT GGC CGC CGG ACT CGA GGT ACA AAT CC-3’)
A7Gly2Ser_rev (SEQ ID NO:22)
(5’-GGT GAT GGC CGC CGG ACT CGA GGT ACA AAT CC-3’)
Gly4SerCys_HindIII (SEQ ID NO:23)
(5’-GGG GGA AGC TTT TAT CAG TGG TGG TGG TGG TGG TGG TGA TGG TGA TGG CAA GAT-3’)
A7Cys4Ser_Nde (SEQ ID NO:24)
(5’-GGA GAT ATA CAAT ATG GGT CTG ATC TCT TTC TCT G-3’)
ATGAAATACC TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC CCAGCCGGCC 60
ATGGCCATGC AAATCTTCGT TAAAACCCTG ACGGGAAAGA CTATCACCCT GGAGGTAGAA 120
CCGTCCGACA CCATCGAAAA TGTCAAAGCT AAAATCCAAG ACAAAGAAGG AATTCCACCT 180
GACCAGCAAC GCCTAGCTTT CGCAGGACGA CAACTAGAGG ACGGGCTCAC CCTGTCTGAC 240
TACAACATCC AAAAAGAATC CACCCTCCAC CTGGCACTCC TCCTGCGGGC C 291
SEQ ID NO: 26
ATGCAAATCT TCGTTAAAAC CCTGACGGGA AAGACTATCA CCCTGGAGGT 50
SEQ ID NO: 27
GGATTTTAGC TTTGACATTT TCGATGGTGT CGGACGGTTC TACCTCCAGG GTG 53
SEQ ID NO: 28
GTCAAAGCTA AAATCCAAGA CAAAGAAGGA ATTCCACCTG ACCAGCAACG CCT 53
SEQ ID NO: 29
GGGTGAGCCC GTCCTCTAGT TGTCGTCCTG CGAAAGCTAG GCGTTGCTGG 50
SEQ ID NO: 30
GACGGGCTCA CCCTGTCTGA CTACAACATC CAAAAAGAAT CCACCCTCCA 50
SEQ ID NO: 31
GAGTGCTCGC AGCAGGAGTG CCAGGTGGAG GGTGGATTC 39
SEQ ID NO: 32
GATATACATA TGCAAATCTT CG 22
SEQ ID NO: 33
GTGGTGCTCG AGTGCTCG 18
SEQ ID NO: 34
CCAGCCGGCC ATGGCCATGN NKATCNNKGT TNNKACCCTG ACGGGAAAGA CTATC 55
SEQ ID NO: 35
CAGGAGGAGT GCCAGGTGGA GMNNMNNMNN MNNMNNGATG TTGTAGTCAG ACAGG 55
SEQ ID NO: 36
GTTATTACTC GCGGCCCAGC CGGCCATGGC CATG 34
SEQ ID NO: 37
GAGTTTTTGT TCGGCCTCGA GGGCCCGCAG GAGGAGTGCC AGGTGGAG 48
Ausuebel, F.M., Brent, R., Kinston, R.E., Moore, D.D., Seidmann, J.G., Smith, J.A. and Struhl, K. (1994): Current protocols in molecular biology. John Wiley & Sons, Inc.
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N. und Bourne, P. E. (2000) The Protein Data Bank. Nucleic Acid Res., 28, 235-242.
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Claims (26)
- 下記成分を含む複合体であり、
SEQ ID NO:9の核酸によりエンコードされるユビキチンに基づく1以上のポリペプチド(I)であって、対応する野生型ポリペプチドと比較して新たに発生または変化させたリガンドに対する特異的結合の結合特性を、それぞれ有し、前記リガンドとの複合体の解離定数K D が10 −5 M以下である前記ポリペプチドと、
それに共有結合的に結合される1以上の機能性成分(II)とを含み、
前記機能性成分(II)が、ポリペプチドおよびタンパク質、有機ポリマーおよび無機ポリマー、核酸、脂質、糖、低分子量物質、ペプチドならびにこれらの物質の誘導体からなる群から選択される少なくとも一つであり、
前記(II)に対する前記(I)のカップリングが、リガンドに特異的に結合するための前記ポリペプチド分子(I)のβシートの表面露出領域の外部領域において、前記ユビキチンにおける2、4、6、62、63、64、65および66番目のアミノ酸からなる群から選択される少なくとも一つの置換により行われ、かつ、(I)への付加的な末端ペプチド融合におけるアミノ酸残基を介して行われるか、または、前記(I)におけるシステインもしくはリジン側鎖を介して、未方向性(未配向性)の形式で、部位特異的もしくは選択的に行われ、
前記(II)に対する前記(I)のカップリング後、全ての成分の機能性が維持されている、複合体。 - 前記リガンドに対する前記(I)の結合表面の外部側鎖が、カップリングに関与する、請求項1記載の複合体。
- 前記ユビキチンに基づくポリペプチド分子(I)において、前記カップリングが、前記ユビキチン分子のリジン残基29および33を介して行われる、請求項1または2記載の複合体。
- 前記(I)に対する付加的な末端ペプチド融合が、1以上のシステイン残基または1以上のリジン残基を含み、これらのアミノ酸残基は、リガンドとの(I)の相互作用に関与するか、または関与しない、請求項1から3のいずれか一項に記載の複合体。
- 前記機能性成分(II)が、ペプチド、ポリペプチドもしくはタンパク質、タンパク質発色団、酵素、免疫グロブリン、免疫グロブリン誘導体、毒素および(I)のポリペプチドからなる群から選択される少なくとも一つである、請求項1から4のいずれか一項に記載の複合体。
- 前記機能性成分(II)が、ポリマー、デキストラン、ポリメタクリル酸、セファロース、アガロース、ポリビニル、ポリスチレン、シリカゲル、セルロースもしくはポリエチレングリコール、およびポリマー誘導体からなる群から選択される少なくとも一つである、請求項1から4のいずれか一項に記載の複合体。
- 前記機能性成分(II)が、低分子量物質、染料、ビオチン、ジオキシゲニン、重金属、キレート剤、放射性同位元素、抗生物質および細胞毒物質からなる群から選択される少なくとも一つである、請求項1から4のいずれか一項に記載の複合体。
- 前記成分(I)が、ポリペプチド、ペプチド、低分子量物質、脂質、糖、核酸、有機ポリマーおよび無機ポリマー、ならびに、これらの物質の誘導体からなる群から選択された少なくとも一つのリガンドに対する新たに発生または変化された特異的結合の結合特性を示す、請求項1から7のいずれか一項に記載の複合体。
- 前記成分(I)が、ポリペプチドまたはタンパク質、免疫グロブリンおよび免疫グロブリン誘導体、血漿から得られるタンパク質、血液凝固因子および阻害剤、成長因子、インターロイキン、サイトカイン、受容体タンパク質、糖化タンパク質、ウイルスタンパク質ならびに細胞表層マーカー、CD14、CD25およびCD34からなる群から選択される少なくとも一つのリガンドに対する新たに発生または変化された特異的結合の結合特性を示す、請求項1から8のいずれか一項に記載の複合体。
- 前記成分(I)が、ペプチド、アフィニティタグ、S−タグ、T7−タグ、His−タグ、Strep−タグ、Myc−タグもしくはFLAG−タグ、および、ウイルス由来ペプチドからなる群から選択される少なくとも一つのリガンドに対する新たに発生または変化された特異的結合の結合特性を示す、請求項1から9のいずれか一項に記載の複合体。
- 前記成分(I)が、低分子量物質、ステロイド、コレステロールおよび、有毒物質、ハロゲン化炭化水素からなる群から選択される少なくとも一つのリガンドに対する新たに発生または変化された特異的結合の結合特性を示す、請求項1から10のいずれか一項に記載の複合体。
- 前記成分(I)が、脂質または脂質誘導体、細菌のリポポリサッカライド、リポソームおよびリポタンパク質からなる群から選択される少なくとも一つのリガンドに対する新たに発生または変化された特異的結合の結合特性を示す、請求項1から11のいずれか一項に記載の複合体。
- 前記成分(II)が、前記(I)と同一の、ユビキチンに基づく1以上のポリペプチドであり、それに共有結合されることによって、(I)のリガンドに対する親和性の向上が、アビディティー効果により達成される、請求項1から12のいずれか一項に記載の複合体。
- 前記成分(II)が、成分(I)が数回共有結合する、ポリペプチド、タンパク質またはポリマーであり、
これによって、前記(I)のリガンドに対する親和性の向上が、アビディティー効果により達成される、請求項1から13のいずれか一項に記載の複合体。 - 前記成分(II)が、ポリペプチドまたはポリマーであり、前記成分(I)に対する共有結合の後、同型の他の複合体に対する共有結合または非共有結合を受け、
これによって、前記(I)のリガンドに対する親和性の向上が、アビディティー効果により達成される、請求項1から14のいずれか一項に記載の複合体。 - 前記成分(I)が、SPU11−3−A1(SEQ ID NO.12および13)の分子のうちの1つによりエンコードされるポリペプチドである、請求項1から15のいずれか一項に記載の複合体。
- 請求項1から16のいずれか一項に記載の複合体を製造する方法であって、既知配列の成分(I)から開始され、下記工程を含む方法。
タンパク質の空間構造の分析による、カップリングに適したアミノ酸残基、リガンドとの(I)の相互作用の表面の外部残基の同定;
適したカップリング試薬によるカップリングパートナーの活性化;
カップリング反応の実行;
複合体の単離;および
複合体の両成分の機能性の検出 - 請求項1から16のいずれか一項に記載の複合体を製造する方法であって、カップリングに適したアミノ酸残基が同定されていない既知配列の成分(I)から開始され、下記工程を含む方法。
置換、挿入または融合によるカップリングに適したアミノ酸残基、リガンドとの(I)の相互作用表面の外部表面に露出される残基の導入;
導入されたアミノ酸残基の露出度の検出;
この方法により変化させた成分(I)の機能性の検出;
適したカップリング試薬によるカップリングパートナーの活性化;
カップリング反応の実行;
複合体の単離;および
複合体の両成分の機能性の検出 - 請求項17または18記載の方法により製造される前記複合体。
- 請求項1から16および19のいずれか一項に記載の複合体を含む診断キット。
- 請求項1から16および19のいずれか一項に記載の複合体、および、薬学的に許容されるキャリアを含む、薬学組成物。
- 前記機能性成分が、膜、ポリマービーズまたはクロマトグラフィー支持材である、請求項1から16および19のいずれか一項に記載の複合体を含む親和性向上のための組成物。
- 診断、治療およびアフィニティークロマトグラフィーに使用するための、請求項1から16および19のいずれか一項に記載の複合体。
- 診断、治療およびアフィニティークロマトグラフィーに使用するための、請求項20記載のキット。
- 診断、治療およびアフィニティークロマトグラフィーに使用するための、請求項21記載の組成物。
- 診断、治療およびアフィニティークロマトグラフィーに使用するための、請求項22記載の組成物。
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US7838629B2 (en) | 2010-11-23 |
CN101084237A (zh) | 2007-12-05 |
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EP1675623A2 (de) | 2006-07-05 |
CA2583009A1 (en) | 2006-04-20 |
ES2414605T3 (es) | 2013-07-22 |
WO2006040129A3 (de) | 2006-06-29 |
CA2583009C (en) | 2012-01-17 |
JP2008516210A (ja) | 2008-05-15 |
US20070248536A1 (en) | 2007-10-25 |
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EP1675623B1 (de) | 2013-03-20 |
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