PMID- 29154787 OWN - NLM STAT- MEDLINE DCOM- 20180105 LR - 20180208 IS - 1096-0309 (Electronic) IS - 0003-2697 (Linking) VI - 540-541 DP - 2018 Jan 1 TI - Functionalization of paramagnetic nanoparticles for protein immobilization and purification. PG - 45-51 LID - S0003-2697(17)30452-9 [pii] LID - 10.1016/j.ab.2017.11.005 [doi] AB - A paramagnetic nanocomposite coated with chitosan and N-(5-Amino-1-carboxy-pentyl) iminodiacetic acid (NTA) that is suitable for protein immobilization applications has been prepared and characterized. The nanoparticle core was synthesized by controlled aggregation of Fe(3)O(4) under alkaline conditions, and Transmission Electron Microscopy revealed a size distribution of 10-50 nm. The nanoparticle core was coated with chitosan and derivatized with glutaraldehyde and NTA, as confirmed by Fourier Transform Infrared Spectroscopy. The final nanoparticles were used as a metal affinity matrix to separate a recombinant polyhistidine-tagged beta-galactosidase from Bacillus subtilis directly from E. coli cell lysates with high purity (>95%). After loading with Ni(2+), nanoparticles demonstrated a binding capacity of 250 mug of a polyhistidine-tagged beta-galactosidase per milligram of support. The immobilized enzyme retained 80% activity after 9 cycles of washing, and the immobilized recombinant protein could be eluted with high purity with imidazole. The applications for these nanomagnetic composites extend beyond protein purification, and can also be used for immobilizing enzymes, where the beta-galactosidase immobilized on the nanomagnetic support was used in multiple cycles of catalytic reactions with no significant loss of catalytic activity. CI - Copyright (c) 2017. Published by Elsevier Inc. FAU - Carneiro, Lara A B C AU - Carneiro LABC AD - Departamento de Bioquimica e Imunologia, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo-USP, Ribeirao Preto, SP CEP 14049-900, Brazil. Electronic address: larabuffoni@gmail.com. FAU - Ward, Richard J AU - Ward RJ AD - Departamento de Quimica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP CEP 14049-901, Brazil. Electronic address: rjward@ffclrp.usp.br. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20171120 PL - United States TA - Anal Biochem JT - Analytical biochemistry JID - 0370535 RN - 0 (Enzymes, Immobilized) RN - 0 (His-His-His-His-His-His) RN - 0 (Imino Acids) RN - 0 (Magnetite Nanoparticles) RN - 0 (Oligopeptides) RN - 0 (Recombinant Proteins) RN - 4QD397987E (Histidine) RN - 9012-76-4 (Chitosan) RN - EC 3.2.1.23 (beta-Galactosidase) RN - XM0M87F357 (Ferrosoferric Oxide) RN - XQM2L81M8Z (iminodiacetic acid) SB - IM MH - Bacillus subtilis/enzymology MH - Chitosan/chemistry MH - *Chromatography, Affinity MH - Enzymes, Immobilized/chemistry/metabolism MH - Ferrosoferric Oxide/chemistry MH - Histidine/chemistry/genetics/metabolism MH - Imino Acids/chemistry MH - Magnetite Nanoparticles/*chemistry MH - Microscopy, Electron, Transmission MH - Oligopeptides/chemistry/genetics/metabolism MH - Particle Size MH - Recombinant Proteins/biosynthesis/chemistry/*isolation & purification MH - Spectroscopy, Fourier Transform Infrared MH - beta-Galactosidase/chemistry/genetics/*metabolism OTO - NOTNLM OT - Chitosan-NTA coating OT - Nanocomposites OT - Protein purification EDAT- 2017/11/21 06:00 MHDA- 2018/01/06 06:00 CRDT- 2017/11/21 06:00 PHST- 2017/08/03 00:00 [received] PHST- 2017/11/08 00:00 [revised] PHST- 2017/11/13 00:00 [accepted] PHST- 2017/11/21 06:00 [pubmed] PHST- 2018/01/06 06:00 [medline] PHST- 2017/11/21 06:00 [entrez] AID - S0003-2697(17)30452-9 [pii] AID - 10.1016/j.ab.2017.11.005 [doi] PST - ppublish SO - Anal Biochem. 2018 Jan 1;540-541:45-51. doi: 10.1016/j.ab.2017.11.005. Epub 2017 Nov 20.