An optimized low-cost protocol for standardized production of iron-free apoferritin nanocages with high protein recovery and suitable conformation for nanotechnological applications

Abstract

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 apoferntin is the first step to use this organic shell as a nanoreactor for different nanotechnological applications. Different protocols have been reported for apoferntin 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 apoferntin 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 apoferntin 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 280 nm. The influence of iron contents on absorbance at 280 nm precludes using this simple spectrophotometnc measure for protein determination in ferritin-iron complexes. Apoferntin produced following our protocol only requires readily-available, cheap and biocompatible reagents, which makes this process standardizable, scalable and applicable to be used for m vivo applications of ferritin derivatives as well as nanotechnological and biotechnological uses.