Development of purification process for dual‐function recombinant human heavy‐chain ferritin by the investigation of genetic modification impact on conformation

Abstract

Ferritin is a promising drug delivery platform and has been functionalized through genetic modifications. This work has designed and expressed a dualfunctional engineered human heavy-chain ferritin (HFn) with the inserted functional peptide PAS and RGDK to extend half-life and improve tumor targeted drug delivery. A facile and cost-effective two-step purification pathway for recombinant HFn was developed. The genetic modification was found to affect HFn conformation, and therefore varied the purification performance. Heat-acid precipitation followed by butyl fast flow hydrophobic interaction chromatography (HIC) has been developed to purify HFn and modified HFns. Nucleic acid removal reached above 99.8% for HFn and modified HFns. However, HFn purity reached above 95% and recovery yield (overall) above 90%, compared with modified HFns purity above 82% and recovery yield (overall) above 58%. It is interesting to find that the inserted functional peptides significantly changed the molecule conformation, where a putative turnover of the E-helix with the inserted functional peptides formed a "flop" conformation, in contrast with the "flip" conformation of HFn. It could be the cause of fragile stability of modified HFns, and therefore less tolerant to heat and acid condition, observed by the lower recovery yield in heat-acid precipitation.

Simple plant‐based production and purification of the assembled human ferritin heavy chain as a nanocarrier for tumor‐targeted drug delivery and bioimaging in cancer therapy

“…The corresponding production costs were ~100,000 € g −1 for plant‐made 90% pure FTH1. This is similar to the purity of commercially available FTH1: 92.5% ± 4.5% (±SD, n = 11) for which a yield of 129 mg L −1 was reported when produced in E. coli (Yin et al, 2021). FTH1 has also been expressed P. pastoris (J.‐L.…”

“…Demand cannot be met by extracting ferritin from animal sources, such as mouse, horse, or pig, reflecting ethical and safety concerns but also cross‐receptor affinity that can hinder clinical translation (Metselaar & Lammers, 2020). Therefore, recombinant FTH1 has been produced in mammalian cells (Origene, 2022) 1 but mostly in microorganisms (Eilert et al, 2012; Guo et al, 2017; Zou et al, 2016) such as Escherichia coli , with yields of up to 129 mg L −1 (Yin et al, 2021). However, this also raises safety concerns because endotoxins are released when extracting recombinant proteins from Gram‐negative bacteria, even though endotoxin‐free strains are now available (Sanchez‐Garcia et al, 2016).…”

Re-engineering the inner surface of ferritin nanocage enables dual drug payloads for synergistic tumor therapy

“…Of note, the encapsulation of the drugs did not influence the size of the nanocarriers, which is consistent with the results of TEM study (Figure 1 D and Figure S1 ). It has been demonstrated that the main secondary structure of HFn is α-helix 47 . In addition, the circular dichroism (CD) spectrum profiles of HFn and ins-FDC nanocarriers were almost the same and contained a pronounced double minimum at around 209 and 222 nm, indicating also the presence of a mainly α-helix secondary structure (Figure 1 G).…”

Rational design of engineered H-ferritin nanoparticles with improved siRNA delivery efficacy across an in vitro model of the mouse BBB

“…Obviously, all positive charges didn't offset the negative charges and there were more negative charges on the inner surface of subunits. 47 The HFn subunit consists of five helices, a, b, c, d, and e: four long α-helices (a, b, c, and d) and one tilted short helix (e) connected by a short loop. The 3D model revealed that helices c, d, and e were present at the inner strands of HFn while helices a and b were present at the outer strands.…”