Comparison of the Immobilization of 6His-Tagged Proteins on Magnetic-Submicron-Particle Functionalized with Ni^{2 +}-NTA and Bis-Sulfone

The screening of ligands in mixtures through magnetic recovery of target-ligand complexes needs site-specific immobilization of 6His-tagged targets on magnetic-submicron-particle. Ni²⁺-NTA-functionalized magnetic-submicron-particle is more readily-available than bis-sulfone-functionalized one. The noncovalent conjugates of 6His-tagged targets and Ni²⁺-NTA-functionalized magnetic-submicron-particle are stable at pH 8.0, but ligand affinities at pH 8.0 may be different from those at pH 7.4. Of full-length human cyclic nucleotide phosphodiesterase 4B2, the inhibition potency of tested neutral compounds was indeed different at pH 7.4 and 8.0. The noncovalent conjugate of 6His-tagged Escherichia coli alkaline phosphatase and Ni²⁺-NTA-functionalized magnetic-submicron-particle released less than 6% of the immobilized enzyme in 3 h at pH 7.4, which enabled the recovery of >90% target-ligand complexes from mixtures of binding reaction. For a 6His-tagged truncated mutant of human cyclic nucleotide phosphodiesterase 4B2 and Ni²⁺-NTA-functionalized magnetic-submicron-particle, the noncovalent conjugate showed similar stability at pH 7.4. After site-specific immobilization on Ni²⁺-NTA-functionalized and bis-sulfone-functionalized magnetic-submicron-particle, these two 6His-tagged enzymes reserved >90% of their original activities. The use of bis-sulfone-functionalized magnetic-submicron-particle caused nonspecific adsorption of those two 6His-tagged enzymes and much longer time for immobilization. Therefore, it was rational to use Ni²⁺-NTA-MSP for site-specific immobilization of 6His-tagged targets to screen ligands in mixtures.