第七章金屬螯合層析一、基本原理

金屬螯合層析

MetalChelateChromatography，MCC又稱為固定化金屬離子吸附層析ImmobilizedMetalIonAdsorptionChromatography,IMAC金屬螯合層析是利用不同蛋白質分子表面所含組氨酸的差別而將其分離純化的一種層析技術。金屬螯合層析的介質通常是在惰性支持物上接有固定化的金屬離子，組氨酸與金屬離子具有螯合作用，對于具有不同組氨酸含量的不同蛋白質分子對于一定金屬離子的親和力大小不同，因而可通過柱層析進行分離。二、金屬螯合劑在Sepharos上接有亞氨基二乙酸Iminodiaceticacid,IDE將氯化鋅或硫酸銅等溶液通過該柱時即可制備出鋅或銅等金屬螯合劑。

IDE

Na+三、影響吸附的因素1.與金屬離子的性質有關Cu2+、Zn2+、Ca2+、Ni2+、Mg2+等2.與樣品的性質有關

His含量不同

His分布位置不同

Buffer的pH及離子強度四、實驗方法20mM,pH8.0PBS+0.5MNaCl+CuSO4平衡Buffer平衡上樣改變pH或離子強度洗脫50mMEDTA再生除去金屬離子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.組氨酸融合系統表達的重組蛋白，