In vitro formation of protein nanoparticle using recombinant human ferritin H and L chains produced from E. coli

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In vitro formation of protein nanoparticle using recombinant human ferritin H and L chains produced from E. coli
H S Ro; Hyun Kyu Park; Min-Gon Kim; Bong Hyun Chung
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 15, no. 2, pp. 254-258
Publication Year
We have conducted in vitro reconstitution study of ferritin from its subunits FerH and FerL. For the reconstitution, FerH was produced from an expression vector construct in Escherichia coli and was purified from a heat-treated cell extract by using one-step column chromatography. FerL was expressed as inclusion bodies. The denatured form of FerL was obtained by a simple washing step of the inclusion bodies with 3 M urea. The reconstitution experiment was conducted with various molar ratios of urea-denatured FerH and FerL to make the ferritin nanoparticle with a controlled composition of FerH and FerL. SDS-PAGE analysis of the reconstituted ferritins revealed that the reconstitution required the presence of more than 40 molar% of FerH in the reconstitution mixture. The assembly of the subunits into the ferritin nanoparticle was confirmed by the presence of spherical particles with diameter of 10 nm by the atomic force microscopic image. Further analysis of the particles by using a transmission electron microscope revealed that the reconstituted particles exhibited different percentages of population with dense iron core. The reconstituted ferritin nanoparticles made with molar ratios of [FerH]/[FerL]=100/0 and 60/40 showed that 80 to 90% of the particles were apoferritin, devoid of iron core. On the contrary, all the particles formed with [FerH]/[FerL]=85/15 were found to contain the iron core. This suggests that although FerH can uptake iron, a minor portion of FerL, not exceeding 40% at most, is required to deposit iron inside the particle.
Korea Soc-Assoc-Inst
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