title: Specific adsorption of histidine-tagged proteins on silica surfaces modified with Ni2+/NTA-derivatized poly(ethylene glycol).
abstract: Silica surfaces modified with nitrilotriacetic acid (NTA)-polyethylene glycol (PEG) derivatives were used to immobilize hexahistidine-tagged green fluorescent protein (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.
authors: School of Biomedical Engineering, School of Chemical Engineering, Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors: Eunah
authors: E
authors: Kang
authors: Jin-Won
authors: JW
authors: Park
authors: Scott J
authors: SJ
authors: McClellan
authors: Jong-Mok
authors: JM
authors: Kim
authors: David P
authors: DP
authors: Holland
authors: Gil U
authors: GU
authors: Lee
authors: Elias I
authors: EI
authors: Franses
authors: Kinam
authors: K
authors: Park
authors: David H
authors: DH
authors: Thompson
mesh_terms: D000327:Adsorption; D015374:Biosensing Techniques; D006639:Histidine / Q000737:chemistry; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry; D015394:Molecular Structure; D009532:Nickel; D009571:Nitrilotriacetic Acid; D011092:Polyethylene Glycols; D011506:Proteins / Q000737:chemistry*; D012822:Silicon Dioxide; D013499:Surface Properties
chemical_list: D008051:Lipid Bilayers; D011506:Proteins; D011092:Polyethylene Glycols; D006639:Histidine; D012822:Silicon Dioxide; D009532:Nickel; D009571:Nitrilotriacetic Acid