Highly-Efficient Purification of Native Polyhistidine-Tagged Proteins by Multivalent NTA-Modified Magnetic Nanoparticles

Abstract

A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His x 6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His x 6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His x 6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His x 6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His x 6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His x 6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.

In Vitro Reconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose

“…To enlarge the complexes beyond the size of a single CipA scaffoldin, spherical nanoparticles were used to reconstitute cellulosome-like particles on an inorganic basis. To achieve this, Coh3-Coh4 miniscaffoldins were chemically bound to the surfaces of 0.11-m ferromagnetic nanoparticles by peptide-coupling chemistry (15,18). The maximum coupling efficiency of protein (79.6 Ϯ 4.5 g mg Ϫ1 of nanoparticles) was achieved at an EDC/sulfo-NHS ratio of 2/5.…”

“…Nanoparticles (20 mg; Estapor microspheres; Merck, Darmstadt, Germany) with a diameter of 0.110 Ϯ 0.007 m, a solid content of 9%, and 497 eq g Ϫ1 of carboxylic surface groups were washed three times in 2 ml activation buffer (50 mM MES, 0.5 M NaCl [pH 6.0]) by separation with a strong NdFeB disc magnet (1.43 Ϯ 0.2 T). The modified particle surface was activated by adding fresh EDC solution [water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; Pierce] and sulfo-NHS solution (Nhydroxysulfosuccinimide; Pierce) to final concentrations of 2 mM and 5 mM, respectively, for 15 min at room temperature (15,18,33). Particles were magnetically separated and washed twice with 2 ml reaction buffer (0.1 M NaH 2 PO 4 , 0.5 M NaCl [pH 7.2]).…”

“…After two washing steps with 2 ml reaction buffer, 10 mg N ␣ ,N ␣ -bis(carboxymethyl)-L-lysine hydrate (NTA) was added and kept at room temperature for 3 h. Particles were washed three times with 2 ml distilled water. One milliliter of 1 M NiSO 4 was added to complex the carboxylate groups of NTA (15). After 5 min, the particles were washed twice with 2 ml distilled water, followed by two washes with 2 ml 50 mM MOPS, 0.1 M NaCl, 5 mM CaCl 2 (pH 6.0).…”

Magnetic Nanoparticles for Early Detection of Cancer by Magnetic Resonance Imaging

“…Several new coating strategies have recently been developed for ferrite nanoparticles, in particular, iron oxide nanoparticles. For example, several functional groups were immobilized on iron oxide nanoparticles with dopamine, 25,26 hydroxamic acid, 27 and 2,3-dimercaptosuccinic acid (DMSA). 24 The DMSA-coated iron oxide nanoparticles were dispersible in water and used for in vivo MRI.…”

Nanotechnology in proteomics

“…Accurate identification of protein subsets from multicomponent solutions is possible from microliters of specimen when magnetic particles are used in conjunction with TOF-MS [32]. In addition to applications involving identification and concentration of low-abundance proteins [33], magnetic nanoparticles have been used to isolate progenitor cells [34] and to detect protein subsets that distinguish healthy patients from those harboring systemic disease [1]. This technology may therefore have a role in screening, diagnosis or monitoring of human disease.…”