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Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents

Paper ID Volume ID Publish Year Pages File Format Full-Text
36059 45119 2006 10 PDF Available
Title
Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents
Abstract

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 concave-downward curves in the Scatchard analysis. The maximum capacities the IMAC adsorbent for the model proteins, declining from 31.79 nmol/g 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 μM−1 for the dimeric epimerase to 50.01 μM−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 μM−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 Langmuir isotherm model.

Keywords
IMAC; Adsorption isotherm; Protein purification; Langmuir–Freundlich
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Effect of number of poly(His) tags on the adsorption of engineered proteins on immobilized metal affinity chromatography adsorbents
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 41, Issue 9, September 2006, Pages 2058–2067
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
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Price was $35.95
You save - $31
Price after discount Only $4.95
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