Preparation and characterization of nickel nanoparticles for binding to his-tag proteins and antigens

Jigna D. Patel, Ronan O'Carra, Julia Jones, Jerold G. Woodward, Russell J. Mumper

Research output: Contribution to journalArticlepeer-review

58 Scopus citations


Purpose. The purpose of these studies was to prepare nanoparticles (NPs) with a small amount of surface-chelated nickel for obtaining enhanced binding of histidine-tagged (his-tag) proteins compared to non-histidine-tagged protein binding to charged nanoparticles. Materials and Methods. NPs were prepared from oil-in-water microemulsion precursors using emulsifying wax, 3 mM Brij 78 and 0.1 mM DOGS-NTA-Ni lipid (referred to as Ni-NPs). The amount of lipid entrapped in the NPs was quantitated by atomic emission spectroscopy (AES). The Ni-NPs were investigated for binding to two his-tag proteins, green fluorescent protein (GFP) and his-tag HIV-1 Gag p24. In vivo studies in mice were carried out to evaluate the immune responses obtained to his-tag Gag p24 bound to Ni-NPs. Results. AES studies demonstrated that approximately 5% of the DOGS-NTA-Ni lipid used was entrapped in the NPs. The optimal binding ratio his-tag GFP and his-tag Gag p24 to Ni-NPs was found to be 1:33.7 and 1:35.4 w/w, respectively. This interaction was stable at 37°C in PBS, pH 7.4 over 4 h and the interaction of his-tag GFP with the Ni-NPs was enhanced compared to control NPs prepared with no Ni on the surface (NTA-NPs). The in vivo studies demonstrated enhanced serum IgG and IgG2a responses to his-tag Gag p24 bound to Ni-NPs compared to protein adjuvanted with Alum or adsorbed on the surface of control NTA-NPs. Conclusions. Ni-NPs can be used to bind strongly to his-tag proteins. This system was demonstrated to have potential applications in vaccine delivery for enhancing immune responses to protein-based vaccines.

Original languageEnglish
Pages (from-to)343-352
Number of pages10
JournalPharmaceutical Research
Issue number2
StatePublished - Feb 2007

Bibliographical note

Funding Information:
Emulsifying wax, comprised of cetyl alcohol and polysorbate 60 (molar ratio of 20:1) and Alum were purchased from Spectrum (New Brunswick, NJ). Phosphate buffered saline, pH 7.4 (PBS), PBS, pH 7.4 with 0.05% Tween 20 (PBS/Tween 20), bovine serum albumin (BSA), and Sepharose CL4B were from Sigma Chemical Co. (St. Louis, MO). Brij 78 was purchased from Uniqema (New Castle, DE). Sheep anti-mouse IgG, peroxidase-linked species specific F(ab_)2 fragment was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). IFN-g ELISA kit, streptavi-dinYhorseradish peroxidase (SvYHRP) and biotinylated rat anti-mouse IgG1 and IgG2a monoclonal antibodies were from BD Biosciences Pharmingen (San Diego, CA). Tetramethylbenzidine (TMB) substrate kit and HisGrabi nickel coated plates were purchased from Pierce (Rockford, IL). Microcon\ YM-100, CentriPlus\ YM-100, 2-mercaptoethanol, certified nickel standard (Claritas\ certified reference material) and nitric acid (trace metal grade) were purchased from Fisher Scientific (Hampton, NH). RPMI 1640, 10% heat-inactivated FBS, HEPES, L-glutamine, penicillin, and streptomycin were from GIBCO (Carlsband, CA). His-tag GFP was purchased from Upstate (Charlottesville, VA). His-tag HIV-1 Gag p24 (his-tag p24) was obtained through the Centralised Facility for AIDS Reagents supported by EU Programme EVA/MRC and the UK Medical Research Council (donated by Dr. I. Jones). 1,2-Dioleoyl-sn-Glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] nickel and ammonium salt, abbreviated DOGSYNTAYNi and DOGSYNTA, respectively, were purchased from Avanti Polar Lipids (Alabastar, AL). Polyvinylidene fluoride (PVDF) membranes, 15% Tris-Hydrochloride (Tris-HCl) SDS-PAGE gels, and Immun-star HRP substrate kit were from Bio-Rad (Hercules, CA). For in vivo studies, female BALB/c mice (6Y8 weeks old) were obtained from Harlan SpragueYDawley Laboratories (Indianapolis, IN).

Funding Information:
This research was funded by NIH-NIAID AI058842 to RJM and JGW. J. Patel was supported, in part, by a Predoctoral fellowship received from the American Foundation for Pharmaceutical Education and the 2005 Dissertation Year Fellowship received from the University of Kentucky Graduate School. The authors would like to thank Tricia Coakley in the Environmental Research and Training Laboratory (ERTL) at the University of Kentucky for her technical assistance in analyzing nanoparticle samples by AES.


  • HIV-1 Gag p24
  • Histidine tagged proteins
  • Nanoparticles
  • Nickel

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)


Dive into the research topics of 'Preparation and characterization of nickel nanoparticles for binding to his-tag proteins and antigens'. Together they form a unique fingerprint.

Cite this