title: Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification.
abstract: A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy.
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
authors.forename: Hu
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. Electronic address: d_xy@tju.edu.cn.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Dong
authors.affiliation: Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China. Electronic address: ysun@tju.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
mesh_terms: D002614:Chelating Agents / Q000378:metabolism*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002846:Chromatography, Affinity; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry; D007098:Imino Acids / Q000737:chemistry*; D007202:Indicators and Reagents; D008670:Metals / Q000737:chemistry*; D008689:Methacrylates / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D017510:Protein Folding; D012685:Sepharose / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D002614:Chelating Agents; C471213:His-His-His-His-His-His; D007098:Imino Acids; D007202:Indicators and Reagents; D008670:Metals; D008689:Methacrylates; D009842:Oligopeptides; C485184:enhanced green fluorescent protein; C528054:glycidyl methacrylate-iminodiacetic acid; D049452:Green Fluorescent Proteins; D006639:Histidine; D012822:Silicon Dioxide; D012685:Sepharose
keywords: Affinity binding; Metal chelator; Nanoparticle; Protein refolding; Surface grafting