Hyperthermostable recombinant human heteropolymer ferritin derived from a novel plasmid design

Abstract

Mammalian ferritins are predominantly heteropolymeric species consisting of 2 structurally similar, but functionally and genetically distinct subunit types, called H (Heavy) and L (Light). The two subunits co-assemble in different H and L ratios to form 24-mer shell-like protein nanocages where thousands of iron atoms can be mineralized inside a hollow cavity. Here, we use differential scanning calorimetry (DSC) to study ferritin stability and understand how various combinations of H and L subunits confer aspects of protein structurefunction relationships. Using a recently engineered plasmid design that enables the synthesis of complex ferritin nanostructures with specific H to L subunit ratios, we show that homopolymer L and heteropolymer L-rich ferritins have a remarkable hyperthermostability (Tm = 115 ± 1 C) compared to their H-ferritin homologues (Tm = 93 ± 1 C). Our data reveal a significant linear correlation between protein thermal stability and the number of L subunits present on the ferritin shell. A strong and unexpected iron-induced protein thermal destabilization effect (ΔT m up to 20 C) is observed. To our knowledge, this is the first report of recombinant human homo-and hetero-polymer ferritins that exhibit surprisingly high dissociation temperatures, the highest among all known ferritin species, including many known hyperthermophilic proteins and enzymes. This extreme thermostability of our L and L-rich ferritins may have great potential for biotechnological applications.

Ferritin microheterogeneity, subunit composition, functional, and physiological implications

“…The pI difference between recombinant and natural human ferritins is partially attributed to the N-terminus that is blocked in the native and free in the recombinant ferritins 41 . Interestingly, a linear plot of the melting temperature (Tm) vs. L-subunits was recently reported 28 , suggesting that ferritin L-subunits exhibit similar effects on the proteins' thermostability and their isoelectric points. Remarkably, out of the available ferritin structures, an improved web-based visualization tool (PDB2PQR) of the Adaptive Poisson-Boltzmann Solver software 42 provided pI values that perfectly matched our experimental values (i.e.…”

“…Protein quantification was performed using the Micro BCA Protein Assay Kit and a Varioskan LUX microplate reader from Thermo Fisher Scientific. Purified recombinant homopolymer and heteropolymer ferritins contained a small iron core (i.e., < 200 ± 50 Fe(III)/ferritin molecule), as determined by an iron reductive mobilization assay 28 .…”

“…Nonetheless, the results of these studies have shown that heteropolymer ferritins have properties that differ significantly from homopolymers 5 . To address this shortcoming, we have engineered a novel ferritin expression system to produce recombinant heteropolymer ferritins with different H to L subunit ratios that mimic those found in various organs and tissues 24 , have characterized their iron uptake and release kinetics, and the structure and morphologies of their iron cores [25][26][27][28] . Our unique plasmid design allowed the synthesis of a full spectrum of heteropolymer ferritins, from H-rich to L-rich ferritins and any combinations in-between, enabling us for the first time to examine structural and functional differences of isoferritin populations [24][25][26][27][28] .…”

Unveiling the stochastic nature of human heteropolymer ferritin self‐assembly mechanism

“…Recombinant human heteropolymer ferritins with different H to L subunit ratios were produced in E. coli Rosetta‐gami B strain our engineered pWUR‐FtH‐TetO‐FtL plasmid and different concentrations of inducers, as described in detail elsewhere (Srivastava et al, 2021; Srivastava et al, 2022; Srivastava et al, 2023). Briefly, transformed cells were induced at 37°C for 4–6 h using 10–1000 μM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG from Sigma Aldrich), 25–1600 ng/mL of anhydrotetracycline (IBA solutions).…”

Sequence-Structure Based Comparison of Structurally Homologous Thermophilic and Mesophilic Polyethylene Terephthalate (PET) Hydrolases

“…Amino acid preference, the ratio of charged versus uncharged amino acids, ionic interactions, codon use, hydrophobicity, and protein surface area are examples of these (Zhou et al 2008). Even though the enzymes used as examples originate from hyperthermophilic sources (Vieille and Zeikus 2001), to date, the publication written two decades ago has become guidance for scientists conducting protein engineering Van Wyk et al 2022;Liu et al 2022;Srivastava et al 2023). Since there, numerous engineering methods to obtain thermostable enzymes or other purposes have been documented (Wang et al 2006;Shirke et al 2018;Khersonsky et al 2018;Noda-Garcia et al 2018;Deng et al 2023).…”