Development and characterization of Ni-NTA-bearing microspheres

For ease of purification, proteins are often expressed with a short affinity sequence of five or six adjacent histidine residues (His-tag). This His-tag binds to the metal of metal chelator complexes such as Ni(2+)-nitrilotriacetic acid (Ni-NTA) or -iminodiacetic acid (Ni-IDA). Chromatography resins bearing covalently attached metal chelator complexes are used widely for the easy affinity purification of His-tagged proteins or peptides. Because Ni-NTA microspheres were not commercially available at the beginning of our studies, we prepared and characterized such microspheres to immobilize His-tagged proteins and study their interactions. Our microspheres are of three types: (a) metal chelator complexes bound covalently to polystyrene microspheres, (b) metal chelator complexes bound covalently to silica microspheres, and (c) lipid-linked metal chelator complexes adsorbed to silica microspheres forming self-assembled bilayer membranes where the metal chelators have lateral mobility.

The microspheres bearing covalently attached Ni-chelator were synthesized by reacting a primary amine-bearing Ni-NTA ligand with carboxy-functionalized microspheres and then loading with Ni(2+). Microspheres with laterally mobile metal chelator were made by incubating glass microspheres with liposomes containing phosphatidylcholine (PC) and the metal chelating lipid 1,2-dioleoyl-sn-glycero-3-[(N (5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]. Binding of a His-tagged enhanced green fluorescent protein (EGFP) was used to characterize these microspheres by flow cytometry for their specificity, sensitivity, capacity and stability.

While all micospheres specifically bind His-tagged proteins, the conditions to achieve this are different for the polystyrene- and silica-based spheres. All three types of microspheres bind His-EGFP with saturation occurring at 30-50 nM and an apparent avidity (concentration of half-maximal binding) of approximately 1 to 2 x 10(-8) M at pH 7.4. Binding of His-EGFP is inhibited by imidazole or ethylene-diaminetetraacetic acid (EDTA). Polystyrene Ni-NTA microspheres showed significant nonspecific binding as measured by binding in the presence of imidazole or EDTA or by binding of fluorescent proteins lacking a His-tag. This nonspecific binding of proteins to and aggregation of polystyrene spheres could only be prevented by the inclusion of low concentrations of Tween 20, but not by including bovine serum albumin (BSA), polyethylene glycols, or polyvinylpyrrolidones as blocking agents. In contrast, silica-based microspheres with covalently attached Ni-NTA or silica microspheres bearing adsorbed bilayers that contain Ni-NTA-lipid showed little nonspecific binding in the presence of BSA. Our results on the stability of immobilization indicate that washing destabilizes the binding of His-tagged proteins to Ni-NTA microspheres. This binding consists of two interactions of different affinities. We also demonstrate that limited multiplexed analysis with differently sized silica microspheres bearing the Ni-NTA-lipid is feasible.

The microspheres described are well suited to selectively immobilize His-tagged proteins to analyze their interactions by flow cytometry. The affinity and kinetic stability of the interaction of His-tagged proteins with Ni-NTA are insufficient to use Ni-NTA microspheres in multiplexed analysis formats where different His-tagged proteins are bound to distinct microspheres. Improvements towards this end (improved chelators and/or improved affinity tags) are critical for extending the use of this method. We are currently working on novel chelators to strengthen the stability of immobilization of His-tagged proteins to surfaces. Such improvements would greatly enhance the analysis of interactions of immobilized His-tagged proteins and could make the development of microsphere-based arrays with His-tagged protein/antibody possible.