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Comparison of the Immobilization of 6His-Tagged Proteins on Magnetic-Submicron-Particle Functionalized with Ni2 +-NTA and Bis-Sulfone

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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.
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Keywords: 6HIS-TAGGED TARGETS; BIS-SULFONE; MAGNETIC-SUBMICRON-PARTICLE; NI2+-NTA; SITE-SPECIFIC IMMOBILIZATION; STABILITY OF CONJUGATE

Document Type: Short Communication

Publication date: June 1, 2015

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  • Nanoscience and Nanotechnology Letters (NNL) is a multidisciplinary peer-reviewed journal consolidating nanoscale research activities in all disciplines of science, engineering and medicine into a single and unique reference source. NNL provides the means for scientists, engineers, medical experts and technocrats to publish original short research articles as communications/letters of important new scientific and technological findings, encompassing the fundamental and applied research in all disciplines of the physical sciences, engineering and medicine.
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