Highly-efficient purification of native polyhistidine-tagged proteins by multivalent NTA-modified magnetic nanoparticles.
A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His x 6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His x 6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His x 6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His x 6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His x 6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His x 6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.
Kim
Jason S
JS
Department of Chemistry and School of Pharmacy and Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Valencia
C Alexander
CA
Liu
Rihe
R
Lin
Wenbin
W
D000818:Animals; D002478:Cells, Cultured; D002614:Chelating Agents; D002846:Chromatography, Affinity; D003598:Cytoskeletal Proteins; D043169:Endostatins / Q000737:chemistry / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008280:Magnetics; D051379:Mice; D053758:Nanoparticles; D009532:Nickel / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000302:isolation & purification
D002614:Chelating Agents; D003598:Cytoskeletal Proteins; D043169:Endostatins; D009687:Nuclear Proteins; D011506:Proteins; C109331:TRIP11 protein, human; D025801:Ubiquitin; C033223:polyhistidine; D006639:Histidine; D009532:Nickel; D043222:Ubiquitin Thiolesterase; C496976:Uchl1 protein, mouse; D009571:Nitrilotriacetic Acid