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.