金属螯合层析MCAC-XP1

第七章金属螯合层析一、基本原理

金属螯合层析

MetalChelateChromatography，MCC又称为固定化金属离子吸附层析ImmobilizedMetalIonAdsorptionChromatography,IMAC金属螯合层析是利用不同蛋白质分子表面所含组氨酸的差别而将其分离纯化的一种层析技术。金属螯合层析的介质通常是在惰性支持物上接有固定化的金属离子，组氨酸与金属离子具有螯合作用，对于具有不同组氨酸含量的不同蛋白质分子对于一定金属离子的亲和力大小不同，因而可通过柱层析进行分离。二、金属螯合剂在Sepharos上接有亚氨基二乙酸Iminodiaceticacid,IDE将氯化锌或硫酸铜等溶液通过该柱时即可制备出锌或铜等金属螯合剂。

IDE

Na+三、影响吸附的因素1.与金属离子的性质有关Cu2+、Zn2+、Ca2+、Ni2+、Mg2+等2.与样品的性质有关

His含量不同

His分布位置不同

Buffer的pH及离子强度四、实验方法20mM,pH8.0PBS+0.5MNaCl+CuSO4平衡Buffer平衡上样改变pH或离子强度洗脱50mMEDTA再生除去金属离子Affinity-taggedfusionproteinsTheaffinity-tagbindstoaspecificligand'Only'thetaggedfusionproteinbindstotheligandBindingusuallypossiblein8MUreaor6MGuHCl(dependsontag)AproteasecleavagesiteallowsthetagtoberemovedafterpurificationPuritytypically>90%inonestepr-ProteinCleavagesiteSpecificligandMatrixAffinitytagTargetproteinAffinitytag LigandGlutathione-S-Transferase(GST) GlutathioneOligo(Histidine) NickelionsEtagsequence Anti-EantibodyZZ (domainBofproteinA) IgGProteinA IgG RecombinantfusionproteinsDeliberatelydesignedforaffinitypurificationNote:Buffersmayinclude8Mureaor6MGuHClwhenpurifyingproteinssolubilisedfrominclusionbodies.ElutionwithCompetingfreeligand

GST-fusionproteinsonGlutathioneSepharoseColumn: GlutathioneSepharose4B,pre-packedBinding: PBS+1%TritonX-100Elution: 5mMGlutathione,50mMTris.HCl,pH8.0PurificationandDetectionof(His)6FusionProteinsr-Prot.HisHisHisNi2+HisHisHisHisNi2+Ni2+Ni2+ElutionwithCompetingfreebindingsubstance

Oligo(His)-fusionproteinsonChelatingSepharoseColumn: HiTrapChelating,Ni2+Binding: 20mMphosphate,0.5MNaCl, 10mMImidazoleElution: Asbindingbufferexcept 500mMImidazoleNote:Buffersmayalsoinclude8Mureaor6MGuHClwhenpurifyingproteinssolubilisedfrominclusionbodies.组氨酸融合系统表达的重组蛋白，