Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.
We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.
Kim
Sung Hoon
SH
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Jeyakumar
M
M
Katzenellenbogen
John A
JA
D015502:Absorptiometry, Photon; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000302:isolation & purification*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005898:Glass; D006639:Histidine / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D053758:Nanoparticles / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009942:Organometallic Compounds / Q000737:chemistry*; D010316:Particle Size; D010777:Photochemistry; D040081:Protein Array Analysis; D012822:Silicon Dioxide / Q000737:chemistry*; D012996:Solutions; D013997:Time Factors
D047628:Estrogen Receptor alpha; D005456:Fluorescent Dyes; D008024:Ligands; D009942:Organometallic Compounds; D012996:Solutions; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D012822:Silicon Dioxide; D009571:Nitrilotriacetic Acid