䜹䝷䝮せ䛷䝅䞁䝥䝹䛺䚸άᛶ䛾䛒䜛䝍䞁䝟䜽㉁䛾✲ᴟ䛾⢭〇ἲ Non-column and simple purification of active proteins expressed as fusions with association-controllable peptide tags ᒸᒣᏛᏛ㝔⮬↛⛉Ꮫ◊✲⛉⏕་⏝ᕤᏛᑓᨷ㻌 㻌 㣕ᯇ㻌 Ꮥṇ ⤌䛘యGST䛾⢭〇 ゎ㞳ྜ䜢ㄪ⠇ྍ⬟䛺䝨䝥䝏䝗䝍䜾䛾Ⓨぢ ⢭〇ᡭ㡰 䝍䜾-䝨䝥䝏䝗 㔞Ⓨ⌧⳦య kDa ཎⅬ ⳦◚○ᾮ 㻌 㻌 㐲ᚰ ୖΎ A A ゎ㞳 97 66 ྜ ᶆⓗ䝍䞁䝟䜽㉁䛻䛴䛡䜜䜀䚸ྜ䛥䛫䜛䚹 45 ỿẊ Ὑί䠖 Ὑίᾮ ᠱ⃮䞉㐲ᚰ A ⣽⬊◚○ A 䜹䝷䝮 Ⓨ䛾ฟⓎⅬ GST 36 29 24 ỿẊ 20 ᢳฟ䞉㐲ᚰ ỿẊ 㧗౯ 㛗㛫 ゎ㞳 BPB ⢭〇GST ᢳฟᾮ ᢏ⾡䛾せ 䝍䜾-䝨䝥䝏䝗 ゎ㞳ྜ⬟ 䝍䜾䝍䞁䝟䜽㉁䛾⁐ゎᛶ 䝍䜾䛝䝍䞁䝟䜽㉁ ᙅሷᇶᛶ 䠄䡚pH9䠅 ୰ᛶ 䠄䡚pH7䠅 ゎ㞳 ⁐ゎ ྜ ỿẊ పሷ⃰ᗘ㻌 (ᩘ10mM) 㧗ሷ⃰ᗘ (ᩘ100mM~) ゎ㞳 => ྍ⁐ᛶ పሷ⃰ᗘ or ᙅ䜰䝹䜹䝸 ᶆⓗ䝍䞁䝟䜽㉁ ྍ⁐ᛶ ྜ ỿẊ ⁐ゎ ⣧ᗘ: ⣙99䠂 ᅇ⋡: ⣙92䠂䠄GSTάᛶ䠅 ゎ㞳 䝍䜾㛫䛾ゎ㞳ྜ ㄪ⠇ྍ⬟ ྜ ྜ䠖ᕧศᏊ =>㻌 ప⁐ゎᛶ ⏕⌮ⓗ䜎䛯䛿 䜔䜔㧗䛔ሷ⃰ᗘ ゎ㞳 ᵓ㐀䞉ᶵ⬟䛿 ಖᣢ䛥䜜䜛 ྜ ỿẊ ⁐ゎ ᐇ ⣽⬊◚○ ⳦◚○ᾮ 㻌 㻌 㐲ᚰ ୖ Ύ ỿẊ Ὑί䠖 Ὑίᾮ ᠱ⃮䞉㐲ᚰ 䠎㔞య ༢㔞య 㔞Ⓨ⌧⳦య kDa ཎⅬ 䜰䝹䝕䝠䝗⬺Ỉ㓝⣲ 䜲䝋䝅䝖䝺䞊䝖䝕䝠䝗䝻䝀䝘䞊䝊 䝩䝇䝩䜶䝜䞊䝹䝢䝹䝡䞁㓟 䜹䝹䝪䜻䝅䜻䝘䞊䝊 ⢭〇ᡭ㡰 kDa ཎⅬ 䠐㔞య kDa ཎⅬ 97 ỿẊ 66 66 ᢳฟ䞉㐲ᚰ ỿẊ ᢳฟᾮ 45 66 45 45 36 36 29 24 29 24 36 29 24 20 20 20 BPB 14 BPB BPB 㛵㐃▱㈈䠖≉㢪2014-185689 䛚ၥྜ䛫ඛ䠖ᒸᒣᏛᏛ㝔⮬↛⛉Ꮫ◊✲⛉㻌 㻌 㣕ᯇ㻌 Ꮥṇ㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㼀㻱㻸㻦㻜㻤㻢㻙㻞㻡㻝㻙㻤㻝㻥㻡㻌㻌㻌㻱㻙㼙㼍㼕㼘㻌㼠㼛㼎㼕㻬㼛㼗㼍㼥㼍㼙㼍㻙㼡㻚㼍㼏㻚㼖㼜㻌 㻌 㻌 Non-column and simple purification of active proteins expressed as fusions with association-controllable peptide tags Takamasa Tobimatsu Graduate School of Natural Science and Technology, Okayama University Starting Point of This Method Purification of Recombinant GST Found a association-controllable peptide tag Procedure E. coli cells Tag-peptide Homogenate centrifuge Supernatant Ppt Wash with buffer/ Washing Centrifuge solution Ppt Extract/ Centrifuge Ppt Expensive, Time consuming A 97 66 A kDa Origin cell rupture A Column A Dissociate Addition of the tags to the protein leads to association. 45 GST 36 Target Protein 29 24 20 BPB Purified Associate Purity: ~ 99䠂 Yield: ~ 92䠂䠄GST activity䠅 Extract (Purified protein) Solubility of Tagged Protein Key Points of This Method Tag Protein Dissociation => High Solubility at low salt conc., or weak alkaline, or surfactant Target Protein Neutral 䠄䡚pH7䠅 Week base 䠄䡚pH9䠅 Tagged Target Protein Dissociation Soluble controllable Tag-Tag interaction Association Precipitate High salt concentration (Several 100mM~) Low salt concentration㻌 (Several 10mM) Association䠖Oligomerized => Low Solubility Dissociation Soluble Association Precipitate Examples Isocitrate dehydrogenase Phospho-enol pyruvate carboxy Procedure E. coli cells Dimer Monomer cell rupture Lactaldehyde dehydrogenase 4 mer Homogenate centrifuge Sup Ppt Wash with buffer/ Washing Centrifuge solution Ppt Extract/ Centrifuge Ppt Extract (Purified protein) kDa kDa Origin Origin kDa Origin 97 66 66 45 66 45 45 36 36 29 24 29 24 36 29 24 20 20 20 BPB 14 BPB Patent pending: Application number 2014-185689 㼀㼍㼗㼍㼙㼍㼟㼍㻌㼀㻻㻮㻵㻹㻭㼀㻿㼁㻌㻳㼞㼍㼐㼡㼍㼠㼑㻌㼟㼏㼔㼛㼛㼘㻌㼛㼒㻌㻺㼍㼠㼡㼞㼍㼘㻌㻿㼏㼕㼑㼚㼏㼑㻌㼍㼚㼐㻌㼀㼑㼏㼔㼚㼛㼘㼛㼓㼥 㻌 㻌 㻌 㻌 㻌 㻌 㻌 㼀㻱㻸㻦㻌㻗㻤㻝㻙㻤㻢㻙㻞㻡㻝㻙㻤㻝㻥㻡㻌㻌㻌㻌㻌㻌 㻌㻱㻙㼙㼍㼕㼘㻦㻌㼠㼛㼎㼕㻬㼛㼗㼍㼥㼍㼙㼍㻙㼡㻚㼍㼏㻚㼖㼜㻌 㻌 㻌 BPB
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