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Selective adsorption of large proteins on highly activated IMAC supports in the presence of high imidazole concentrations: Purification, reversible immobilization and stabilization of thermophilic α- and β-galactosidases

Paper ID Volume ID Publish Year Pages File Format Full-Text
18284 42717 2007 7 PDF Available
Title
Selective adsorption of large proteins on highly activated IMAC supports in the presence of high imidazole concentrations: Purification, reversible immobilization and stabilization of thermophilic α- and β-galactosidases
Abstract

Natural proteins mainly adsorb on immobilized metal ion affinity chromatography (IMAC) supports via multipoint interactions between several His residues placed on the protein surface and different chelate moieties located on the support surface. The adsorption of large proteins on highly activated IMAC supports may involve many multi-interactions yielding a very strong adsorption. In fact, multimeric α- and β-galactosidases from Thermus sp. T2 hardly may be desorbed from highly activated IMAC supports, even in the presence of 1 M imidazole. In the presence of 50 mM of imidazole, these very large proteins can be adsorbed on these supports while the medium-small proteins hardly adsorb even on highly activated supports. In this way a very simple purification and reversible immobilization of these large proteins cloned on Escherichia coli can be performed by first heating of the crude preparation which leaves as the only large protein the thermophilic one. Interestingly, β-galactosidase from Thermus sp. T2 is 10-fold more stable than the native enzyme when incubated at 70 °C and pH 7.0. The involvement of large areas of these multimeric enzymes in the adsorption process may promote a multi-subunit adsorption with stabilizing effects. These immobilized-stabilized enzymes may be desorbed away from the support/the reactor after inactivation and a fresh solution of enzyme can be purified, immobilized and stabilized again.

Keywords
Multipoint adsorption; Selective adsorption; Selective immobilization; Multimeric proteins; Thermophilic proteins
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Selective adsorption of large proteins on highly activated IMAC supports in the presence of high imidazole concentrations: Purification, reversible immobilization and stabilization of thermophilic α- and β-galactosidases
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 2, 4 January 2007, Pages 242–248
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us