Journal
JOURNAL OF NANOPARTICLE RESEARCH
Volume 13, Issue 6, Pages 2475-2484Publisher
SPRINGER
DOI: 10.1007/s11051-010-0140-y
Keywords
Magnetic nanoparticles; Protein separation; Immobilized metal ion affinity; 6x His-tag protein
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Funding
- Department of Biotechnology, Govt. of India
- CSIR, Govt. of India
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The aim of this study was to develop a simple, cheap, and rapid method for purification of His-tag recombinant proteins with high yields. The new immobilized metal ion affinity adsorbent containing superparamagnetic nanoparticles and hydrophilic resins are proposed here to improve the purification of His-tagged recombinant proteins. In this report, we have described the preparation of nanosized superparamagnetic nanoparticles (Fe3O4) which were prepared by chemical precipitation method followed by surface modification using phosphonomethyl iminodiacetic acid. The stable surface functionalized nanoparticles were further linked with Ni2+ for purification of 6x His-tagged proteins. The phosphonate group of the N-phosphonomethyl iminodiacetic acid ligand acts as a surface anchoring agent on magnetite nanoparticles and the remaining free -COOH groups outside for binding with Ni2+ ions. The nanoparticles were approximately 6-8 nm in size and were stable and had negligible non-specific binding for protein. The proteins were purified within 1 h and observed on sodium dodecyl sulfate-polyacrylamide electrophoresis gel.
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