433
‫଻֦ٸ‬ͺεΠȜΏΑ͈ည൵͂௶೰ͅ
ŇŢŴࠐႹࡄ‫ݪ‬ဥȁͺεΠȜΏΑည൵ͺΛΓͼ
ᧄ⵾ຠ䈲䇮Death䊧䉶䊒䉺䊷䈪䈅䉎Fas䋨CD95䋩䈫䇮䈠䈱䊥䉧䊮䊄䈪䈅䉎FasL䋨CD95L䋩䈫䈱⚿ว䉕೑↪䈚䈢䇮䉝䊘䊃䊷䉲䉴⺃ዉ
䉝䉾䉶䉟⹜⮎䈪䈜䇯IBA␠䈏଻᦭䈜䉎䇮Strep-tag /Strep-Tactin 䉺䉫ᛛⴚ䉕೑↪䈚䈩䈍䉍䇮㊁↢ဳFasL䉕೑↪䈚䈢⺃ዉ䉋䉍䉅
㜞ല₸䈪䉝䊘䊃䊷䉲䉴䉕⺃ዉ䈜䉎䈖䈫䈏䈪䈐䉁䈜䇯
R
․˴㐳
R
٨ᵤᵿᶑᵪểᵤᵿᶑểỉႺ੗ႎễኽӳửย‫ܭ‬ẴỦỉỂỴἯἚὊἉἋử‫ܭ‬᣽҄ỂẨỦ
٨ᵲᵒᵋᵤᶍᶊᶂᶍᶌᴾɤ᣽˳ἛἳỶὅỉ੔ဇỂјௐႎỂϐྵࣱỉ᭗ẟỴἯἚὊἉἋᛔ‫ݰ‬ầӧᏡ
٨෩ᵋ෩ίᵦᶍᶋᶍᶅᶃᶌᶃᶍᶓᶑὸỴἕἍỶỂỊẰỤỆјྙỉợẟỴἯἚὊἉἋỉᛔ‫ݰ‬ầӧᏡ
٨‫׍‬ᵋ෩ίᵦᶃᶒᶃᶐᶍᶅᶃᶌᶃᶍᶓᶑὸỴἕἍỶỊỴἯἚὊἉἋ᧹ܹдỉἡỶἋἽὊἩἕἚἋἁἼὊἝὅἂầӧᏡ
ේ˴ℂ
Spacer
184
N
⚦⢩⴫㕙䈱
Fas䈫⚿ว
ਃ㊂૕ൻ
䊐䉤䊷䊦䊂䉞䊮䉫
C
RBD
183
T4-Foldon䊄䊜䉟䊮
Spacer Strep-t ag ®
250 260
SA
142
Strep-tag ®
T4-Foldon
Fas
RBD
281
⊒⃻䈚䈢Ⲣว䉺䊮䊌䉪⾰
න㊂૕
ਃ㊂૕
䋨⚂30kDa䋩
䋨⚂90kDa䋩
ᮡ⊛⚦⢩
FasL-Strep䈲㊁↢ဳFasL䈱⚦⢩ᄖ䊄䊜䉟䊮䈱ਥⷐㇱಽ䈮䊋䉪䊁䊥䉥䊐䉜䊷䉳T4䈱fibritin䉺䊮䊌䉪⾰䈱Cᧃ
┵䊄䊜䉟䊮䉕Ⲣว䈚䈢䉺䊮䊌䉪⾰䈪䈜䇯FasL-Strep䈲T4-Foldon䊄䊜䉟䊮䈱ലᨐ䈪䇮⊒⃻ᓟ䈜䉂䉇䈎䈮቟ቯ䈭
ਃ㊂૕䈮䈭䉍䉁䈜㶎䇯䈠䈚䈩ᮡ⊛⚦⢩䈱⚦⢩⤑䈮ሽ࿷䈜䉎Fas䈮⚿ว䈚䇮䉝䊘䊃䊷䉲䉴䉕ല₸䉋䈒⺃ዉ䈚䉁䈜䇯
̪Tao, Y. et al. Structure. 5(6), 789
⵾ຠㆬᛯ
FasL-Strep䉝䉾䉶䉟䈲቟ቯᕈ䈏㜞䈒䇮ౣ⃻ᕈ䈮ఝ䉏䈩䈇䉁䈜䇯䉁䈢䇮ቯ㊂⸃ᨆ䈏น⢻䈪䈜䇯᛫䉝䊘䊃䊷
䉲䉴ಽሶ䈱䊊䉟䉴䊦䊷䊒䉾䊃䈭䉴䉪䊥䊷䊆䊮䉫䈭䈬䈮䈍૶䈇䈒䈣䈘䈇䇯
ʼnŦŵŦųŰŨŦůŦŰŶŴġŇŢŴōĮġġġġġġġġġġłűŰűŵŰŴŪŴġłŴŴŢź
⵾ຠ⺑᣿
Strep-Tactin 䉕੺䈚䈩FasL-Strep䉕࿕ቯൻ䈚䈢䊙䉟䉪䊨䊒䊧䊷䊃䉕↪䈇䇮⚦⢩⴫㕙䈱Fas䉕ೝỗ䈚䈩䇮䉝䊘䊃䊷
䉲䉴䉕⺃ዉ䈚ቯ㊂ൻ䈚䉁䈜䇯
R
䂓㩷෻ᔕ䈱᭎⇛
䂓ᠲ૞ᚻ㗅
Strep-Tactin R coated microplate
Strep-Tactin®
Strep-tag ®
T4-Foldon䊄䊜䉟䊮
FasL-Strep
FasL
(1) Strep-Tactin㵘㶎䉕䉮䊷䊃䈚䈢䊙䉟䉪䊨䊒䊧䊷䊃䈮
㵘㵘㩷FasL-Strep䉕ᷝട
(2) (1)䈮ᮡ⊛⚦⢩䉕ᷝട
(3) ᮡ⊛⚦⢩䈱Fas䈮䇮Strep-tag㵘/Strep-Tactin㵘㑆䈱
㩷㩷㩷㩷㩷⚿ว䈮䉋䈦䈩䊙䉟䉪䊨䊒䊧䊷䊃䈮࿕ቯൻ䈘䉏䈢FasLStrep䈏⚿ว䈚䇮䉝䊘䊃䊷䉲䉴䈏ᆎ䉁䉎
(4) 䉝䊘䊃䊷䉲䉴ᵴᕈ䈲䉦䉴䊌䊷䉷3䈅䉎䈇䈲7䈱ᵴᕈ䉕
㩷㩷㩷㩷㩷᷹ቯ䈚䈩ᬌ಴
R
R
R
Fas
ᮡ⊛⚦⢩
㶎Strep-Tactin㵘䈲䉴䊃䊧䊒䊃䉝䊎䉳䊮䉕ᡷᄌ䈚䈢䉺䊮䊌䉪⾰䈱྾㊂૕䈪䈜䇯Strep-tag II/Strep-Tactin㵘㑆䈱ⷫ๺ᕈ䈲䇮
˴Strep-tag II/䉴䊃䊧䊒䊃䉝䊎䉳䊮䈱100୚ㄭ䈒ᒝജ䈪䈜䇯
R
R
R
R
ʼnŰŮŰŨŦůŦŰŶŴġŇŢŴōĮġġġġġġġġġġłűŰűŵŰŴŪŴġłŴŴŢź
⵾ຠ⺑᣿
Homogeneous䉝䉾䉶䉟䈪䈲䇮䊙䉡䉴䊝䊉䉪䊨䊷䊅䊦᛫૕䈪䈅䉎StrepMAB-Immo䈮FasL-Strep䉕䉪䊨䉴䊥䊮䉪
䈘䈞䈩䇮䉝䊘䊃䊷䉲䉴䉕⺃ዉ䈚䉁䈜䇯StrepMAB-Immo䉕↪䈇䉎䈖䈫䈪FasL-Strep䈏⚦⢩⴫㕙䈱Fas䈮ല₸䉋䈒
⚿ว䈜䉎䈢䉄䇮䉝䊘䊃䊷䉲䉴⺃ዉᵴᕈ䈏Ⴧ᏷䈘䉏䉁䈜䇯
䂓㩷෻ᔕ䈱᭎⇛
StrepMAB-Immo
FasL-Strep
Fas
ᮡ⊛⚦⢩
100
૶↪଀
Relative Caspase Activity
Relative Caspase Activity
120
100
80
Fas-Fc
60
40
Titration in presence of
250 ng/ml FasL-Strep
20
FasL-Strep
80
FasL-Strep +
StrepMAB-Immo
60
40
20
0
0
0
1
10
100
1000
10000
1
10
100
1000
FasL-Strep [ng/ml]
Fas-Fc [ng/ml]
Fas-Fc䋨਄࿑ෳᾖ䋩䈱᦭ലỚᐲ䉕᳿ቯ䈜䉎䈢䉄䈮䇮
FasL-Strep䉕↪䈇䈩䉝䉾䉶䉟䉕ⴕ䈇䉁䈚䈢䇯
Fas-Fc䈱Ớᐲ䈱਄᣹䈮઻䈦䈩䉦䉴䊌䊷䉷ᵴᕈ䈏ૐਅ
䈜䉎䈖䈫䈎䉌䇮ᧄ⵾ຠ䈏FasL⚻〝䉕੺䈚䈢䉝䊘䊃䊷䉲
䉴䉕⺃ዉ䈚䈩䈇䉎䈖䈫䈏䉒䈎䉍䉁䈜䇯
FasL-Strepන⁛䈪૶↪䈚䈢䈫䈐䈫FasL-Strep䈮
StrepMAB-Immo䉕૬↪䈚䈢䈫䈐䈱䉝䊘䊃䊷䉲䉴ᵴ
ᕈ䉕Ყセ䈚䉁䈚䈢䇯
䊝䊉䉪䊨䊷䊅䊦᛫૕䈪䈅䉎StrepMAB-Immo䈮䉋䈦
䈩䉝䊘䊃䊷䉲䉴ᵴᕈ䈏Ⴧ᏷䈘䉏䉎䈖䈫䈏⏕⹺䈘䉏䈩
䈇䉁䈜䇯
᷹ቯ᧦ઙ
Jurkat A3 cell䈮FasL-Strep䉕ട䈋䈩䇮37㷄䈪3ᤨ㑆䉟䊮䉨䊠䊔䊷䉲䊢䊮㸢⚦⢩䉕ṁ⸃䈚䈩Ac-DEVDAFC䋨Biomol␠⵾䋩䉕ട䈋䇮䉝䊘䊃䊷䉲䉴䉕ᬌ಴䇯
ଔᩰ⴫
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㵘
㵘
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