期刊
NANOSCIENCE AND NANOTECHNOLOGY LETTERS
卷 7, 期 6, 页码 486-494出版社
AMER SCIENTIFIC PUBLISHERS
DOI: 10.1166/nnl.2015.1996
关键词
Site-Specific Immobilization; 6His-Tagged Targets; Magnetic-Submicron-Particle; Ni2+-NTA; Bis-Sulfone; Stability of Conjugate
资金
- National Natural Science Foundation of China [30472139]
- Natural Sciences Foundation of CQ [CSTC2012JJA0057]
- Education Ministry of China [20125503110007]
- Sciences and Technology Commission of Yuzhong District of Chongqing [20130135]
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-subnnicron-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 coil alkaline phosphatase and Ni2+-NTA-functionalized magnetic-subnnicron-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-subnnicron-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-subnnicron-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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