Specific adsorption of histidine-tagged proteins on silica surfaces modified with Ni2+/NTA-derivatized poly(ethylene glycol)

초록

Silica surfaces modified with nitrilotriacetic acid (NTA)-polyethylene glycol (PEG) derivatives were used to immobilize hexahistidine-tagged green fluorescent protein (His(6)-GFP), biotin/streptavidin-AlexaFluor555 (His(6)-biotin/SA-AF), and gramicidin A-containing vesicles (His(6)-gA). Three types of surface-reactive PEG derivatives-NTA-PEG3400-Si(OMe)(3), NTA-PEG3400-vinylsulfone, and mPEG5000-Si(OMe)(3) (control)-were grafted onto silica and tested for their ability to capture His(6)-tag species via His(6)/Ni2+/NTA chelation. The composition and thicknesses of the PEG-modified surfaces were characterized using X-ray photoelectron spectroscopy, contact angle, and ellipsometry. Protein capture efficiencies of the NTA-PEG-grafted surfaces were evaluated by measuring fluorescence intensities of these surfaces after exposure to His(6)-tag species. XPS and ellipsometry data indicate that surface adsorption occurs via specific interactions between the His(6)-tag and the Ni2+/NTA-PEG-grafted surface. Protein immobilization was most effective for NTA-PEG3400-Si(OMe)(3)-modified surfaces, with maximal areal densities achieved at 45 pmol/cm(2) for His(6)-GFP and 95 fmol/cm(2) for His(6)-biotin/SA-AF. Lipid vesicles containing His(6)-gA in a 1:375 gA/lipid ratio could also be immobilized on Ni2+/NTA-PEG3400-Si(OMe)(3)-modified surfaces at 0.5 mM total lipid. Our results suggest that NTA-PEG-Si(OMe)(3) conjugates may be useful tools for immobilizing His(6)-tag proteins on solid surfaces to produce protein-functionalized surfaces.

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