第七章金屬螯合層析一、基本原理

金屬螯合層析

MetalChelateChromatography，MCC又稱(chēng)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(zhì)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(shù)。金屬螯合層析的介質(zhì)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(duì)于具有不同組氨酸含量的不同蛋白質(zhì)分子對(duì)于一定金屬離子的親和力大小不同，因而可通過(guò)柱層析進(jìn)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(guò)該柱時(shí)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(zhì)有關(guān)Cu2+、Zn2+、Ca2+、Ni2+、Mg2+等2.與樣品的性質(zhì)有關(guān)

His含量不同

His分布位置不同

Buffer的pH及離子強(qiáng)度四、實(shí)驗(yàn)方法20mM,pH8.0PBS+0.5MNaCl+CuSO4平衡Buffer平衡上樣改變pH或離子強(qiáng)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(tǒng)表達(dá)的重組蛋白，