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|標題:||Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents||作者:||Tsai, S.Y.
|關鍵字:||IMAC;adsorption isotherm;protein purification;Langmuir-Freundlich;performance liquid-chromatography;aqueous 2-phase systems;containing;cytochrome-c;ion affinity;substrate-specificity;crystal-structure;varying particle;structural basis;ligand density;amino-acids||Project:||Process Biochemistry||期刊/報告no：:||Process Biochemistry, Volume 41, Issue 9, Page(s) 2058-2067.||摘要:||
The equilibrium adsorption of four homo-oligomeric recombinant proteins containing up to eight poly(histidine) affinity tags on a silica-based Cu(II)-IDA immobilized metal affinity chromatography (IMAC) adsorbent is reported in this study. The equilibrium adsorption data are well fitted with the three-parameter Langmuir-Freundlich isotherm model, indicating the presence of positive cooperativity for the adsorption of these model proteins and the degree of cooperativity seems to increase with the number of poly(His) tags. This phenomenon is also supported by the concavedownward curves in the Scatchard analysis. The maximum capacities the IMAC adsorbent for the model proteins, declining from 31.79 nmoVg gel for 88 kDa epimerase to 21.50 nmol/g gel for 320 kDa racemase, are in principle affected by the size of the molecule, and the binding strengths, expressed in term of association constants Ka increasing from 1.47 mu M-1 for the dimeric epimerase to 50.01 mu M-1 for the octameric racemase, are dictated by the number of affinity tags. Nevertheless, some structure-specific interactions observed among native N-carbamoylase molecules may promote multilayer adsorption and thus lead to a much higher adsorption capacity, 715 nmol/g gel and a lower compounded binding affinity, 0.42 mu M-1. The observed positive cooperativity for the model proteins and the structure-specific interactions among N-carbamoylase molecules are eliminated under denaturing conditions, resulting in a homogeneous adsorption well fitted by the Langnmir isotherm model. (c) 2006 Elsevier Ltd. All rights reserved.
|Appears in Collections:||化學工程學系所|
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