Specific Adsorption of Histidine-Tagged Proteins on Silica Surfaces Modified with Ni2+:NTA-Derivatized Poly(Ethylene Glycol)

摘要

Silica surfaces modified with nitrilotriacetic acid (NTA)-polyethylene glycol (PEG) derivatives were used for immobilizing hexahistidine-tagged green fluorescent oprotein (his6-GFP), biotin/streptavidin-AlexaFluor555 (his6-biotin/SA-AF) and gramicidin A-containing vesicles (his6-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 his6-tag species via his6: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 his6-tag species. XPS and ellipsometry data indicate that surface adsorption occurs via specific interactions between the his6-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/cm² for his6-GFP and 95 fmol/cm² for his6-biotin/SA-AF. Lipid vesicles containing his6-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 his6-tag proteins on solid surfaces to produce protein-functionalized surfaces.