An optimized low-cost protocol for standardized production of iron-free apoferritin nanocages with high protein recovery and suitable conformation for nanotechnological applications.
J Inorg Biochem
; 183: 184-190, 2018 06.
Article
em En
| MEDLINE
| ID: mdl-29279245
Ferritin is a globular protein that consists of 24 subunits forming a hollow nanocage structure that naturally stores iron oxyhydroxides. Elimination of iron atoms to obtain the empty protein called apoferritin is the first step to use this organic shell as a nanoreactor for different nanotechnological applications. Different protocols have been reported for apoferritin formation, but some are time consuming, others are difficult to reproduce and protein recovery yields are seldom reported. Here we tested several protocols and performed a complete material characterization of the apoferritin products using size exclusion chromatography, UV-vis spectroscopy, inductively coupled plasma optical emission spectrometry and dynamic light scattering. Our best method removes more than 99% of the iron from loaded holoferritin, recovering 70-80% of the original protein as monomeric apoferritin nanocages. Our work shows that pH conditions of the reduction step and the presence and nature of chelating agents affect the efficiency of iron removal. Furthermore, process conditions also seem to have an influence on the monomer:aggregate proportion present in the product. We also demonstrate that iron contents markedly increase ferritin absorbance at 280nm. The influence of iron contents on absorbance at 280nm precludes using this simple spectrophotometric measure for protein determination in ferritiniron complexes. Apoferritin produced following our protocol only requires readily-available, cheap and biocompatible reagents, which makes this process standardizable, scalable and applicable to be used for in vivo applications of ferritin derivatives as well as nanotechnological and biotechnological uses.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Apoferritinas
/
Nanotecnologia
/
Nanoestruturas
Tipo de estudo:
Health_economic_evaluation
Idioma:
En
Revista:
J Inorg Biochem
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Chile
País de publicação:
Estados Unidos