Functionalization of paramagnetic nanoparticles for protein immobilization and purification

Autor(es)

Carneiro, Lara A. B. C. ^[1] ; Ward, Richard J. ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, BR-14049900 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, Ave Bandeirantes 3900, BR-14049901 Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Analytical Biochemistry; v. 540, p. 45-51, JAN 1 2018.
• Citações Web of Science: 5

Resumo

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 Fe3O4 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. colt cell lysates with high purity (> 95%). After loading with Ni2+, nanoparticles demonstrated a binding capacity of 250 jig 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. (AU)

• Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
• Beneficiário: Richard John Ward
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 16/15708-7 - Caracterização de um novo produto de hidrólise do xiloglucano resultante da ação sinérgica entre uma mistura de beta-galactosidase/alfa-xilosidase
• Beneficiário: Lara Aparecida Buffoni de Campos Carneiro
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
• Processo FAPESP: 12/24147-8 - Desenho racional de enzimas multifuncionais para despolimerização do material lignocelulósico
• Beneficiário: Lara Aparecida Buffoni de Campos Carneiro
• Modalidade de apoio: Bolsas no Brasil - Doutorado