Reversible and oriented immobilization of histidine-tagged protein on silica gel characterized by frontal analysis

RSC Advances Pub Date: 2015-03-06 DOI: 10.1039/C5RA01012H

Abstract

This approach utilized N,N′-bis(carboxymethyl)-L-lysine (ANTA) coordinated to bivalent metal cation Ni2+, leaving free coordination sites for the reversible binding of gene recombinant histidine-tagged β2-adrenoceptor onto macropore silica. The amount of transient metal nickel ion on the support was determined by atomic absorption spectrophotometry. The novel protein oriented immobilization β2-AR column was evaluated by five β2-adrenoceptor agonists, applying frontal analysis. The association equilibrium constant for ligands on the column was 1.98 × 104 M?1 for salbutamol, 3.43 × 104 M?1 for clenbuterol, 2.09 × 104 M?1 for tulobuterol, 1.84 × 104 M?1 for terbutaline, 1.71 × 104 M?1 for methoxyphenamine and corresponding concentrations at binding sites were 7.46 × 10?6 M, 1.82 × 10?5 M, 2.16 × 10?5 M, 8.29 × 10?6 M and 3.88 × 10?5 M, respectively. The results obtained from breakthrough and nonlinear fitting indicated that all the drugs have a single binding site on the β2-adrenoceptor column. The present combined histidine-tagged protein method was reliable and exact in revealing interactions between receptor and drugs.

Graphical abstract: Reversible and oriented immobilization of histidine-tagged protein on silica gel characterized by frontal analysis
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