Heavy chain human ferritin

This part is the heavy ferritin subunit from human ferritin, inspired by P02794 [UniParc]. Ferritin is ubiquitous across prokaryotic and eukaryotic systems and is used to buffer intracellular iron. This part, along with the light ferritin subunit, form a 24 multimeric iron sequestering nanoparticle (Chasteen et al., 1991). The difference between light ferritin is that this chain contains a ferroxidase centre. Protein domains which orient toward the core of ferritin molecules cause the oxidation of intracellular iron from Fe^2+ to Fe^3+ to initiate formation of a ferrihydrite core. (Chasteen et al., 1999). These nanoparticles are robust, remain stable at extreme pHs, and withstand temperature variations (Kim et al., 1999).

Ferritin's utility in iGEM

Ferritin as a nanoparticle is interesting for other iGEM teams for two reasons. Firstly, its iron core can be replaced with other compounds to serve different functions. The iGEM Calgary 2013 demonstrated this by chemically modifying recombinant ferritin's iron core into a robust colourmetric reporter. Other intriguing applications include making ferritin’s iron core magnetically active as magnetoferritin (Jordan et al. 2013), using ferritin as a nanocage for other metals, or the incorporation of other reporters such as quantum dots (Naito et al. 2013) (Figure 2).

Ferritin Core Modulation — **Figure 2.** Chemically modifying the iron core of ferritin allows ferritin to be moulded to fit a wide magnitude of applications. Additionally the ferritin subunits can act as a nanocage to encapsulate completely new cores.

Secondly, the ferritin nanoparticle is useful for iGEM teams as a self-assembling and spherical protein scaffold. Each of the 24 subunits forming ferritin can be fused to proteins of interest, such that when the nanoparticle assembles, proteins surround the ferritin sphere (Kim et al., 2011). The iGEM Calgary 2013 team demonstrated this by binding DNA sensing proteins, TALEs, as part of their FerriTALE sensor. The Calgary team also constructed ferritin subunits with a coiled-coil linker system so that other teams can scaffold proteins to E-coil ferritin (BBa_K1189018, BBa_K1189019, BBa_K1189020, BBa_K1189037). See Figure 3 for a demonstration of these applications.

FerriTALE Scaffold Modularity — **Figure 3.** Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.

References

Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.

Clavijo Jordan, V., Caplan, M. R., & Bennett, K. M. (2010). Simplified synthesis and relaxometry of magnetoferritin for magnetic resonance imaging. Magnetic Resonance in Medicine, 64(5), 1260-1266.

Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. Analytical chemistry, 83(15), 5834-5843.

Naito, M., Iwahori, K., Miura, A., Yamane, M., & Yamashita, I. (2010). Circularly polarized luminescent CdS quantum dots prepared in a protein nanocage. Angewandte Chemie International Edition, 49(39), 7006-7009.

GreatBay_SCIE 2022's Characterisation

Magnetic recycling

A novel design feature of our engineered E.coli was the inclusion of intracellular ferritin expression. The Fe2+ ions in ferritin allows the E.coli cells displaying cellulosome complex to be attracted by strong magnets, therefore enabling the magnetic recycling of cellulosomes to be reused.

We’ve constructed three ferritin plasmids: the ferritin wild type (Pfuferritin), the existing part (BBa_K1189065), and the wild type ferritin fused with Nb3 (Pfuferritin-Nb3) (Fig.1A). All three vectors were transformed and cultured for IPTG-inducible expression. The target proteins of all three ferritins were detected in the whole cell and supernatant samples of SDS-page (Fig.1B).

To further prove that the host E.coli cells carrying ferritin is magnetically attractive, we transformed prha-mRFP1 plasmids into competent cells containing Pfuferritin and BBa_K1189065 respectively. The co-expression of ferritin and RFP allowed magnetic recycling results to be better visualized. In both ferritin wild type and ferritin part, aggregation of red fluorescence were found near the strong magnets, proving the feasibility of magnetic recycling system (Fig.1C).

Figure 1: Fig.1 Ferritin expression and magnetic recycling. (A) Genetic circuit construction for three types of ferritin: ferritin wild type (PfuFerritin), IGEM existing ferritin part (BBa_K1189025), and ferritin-Nb3 (PfuFerritin-Nb3) adapted for surface display system. (B) SDS-page analysis of PfuFerritin-Nb3, BBa_K1189025, Ferritin wild type (WT) respectively. (C) Magnetic recycling was conducted with Ferritin control group, RFP control group, BBa_K1189025-RFP, and Ferritin WT-RFP. Apparent aggregation of RFP fluorescence shown in cells co-expressing ferritin and RFP verified the ability for ferritin to be attracted by strong magnets.

Contribution (Waterloo iGEM 2020)

Summary: While the heavy and light chain human ferritin subunits have been added to the iGEM parts registry with adequate description of their function, we wanted to compare them more thoroughly. Therefore, our discussion includes both chains’ stability and ability to uptake iron.

Documentation: Human ferritin heteropolymers can be composed with varying ratios of heavy (H) and light (L) chains, and different forms exist naturally depending on the tissue examined (Boyd et al., 1985). However, the knowledge of the two chains is not sufficient enough to predict the properties of any given heteropolymer.

Homopolymers composed of strictly light or heavy chains can be synthesized, which can be advantageous as heavy and light chains have different properties: 1. H-rich ferritins turn over more rapidly than L-rich ferritins 2. H-rich ferritins accumulate in iron-rich tissues 3. L-rich ferritins take up and release iron more rapidly than L-rich ferritins

Within the shell, H and L subunits are interchangeable as the key residues are conserved.

L subunits contain 15 hydrophilic iron binding residues, while only 7 of these exist in H subunits. This results in a reduced ability to nucleate iron in H subunits, causing faster iron uptake and release as well as less iron accumulation in H-rich ferritins.

Ferritin’s uptake of iron is either driven by H-chain ferroxidase activity (Levi et al., 1988) or driven by iron autoxidation which occurs in the absence of H-chains.

Stability:

In vitro, H ferritin oxidizes iron at a much faster rate than L ferritin because of the presence of a particular ferroxidase centre in H ferritin that is absent in L ferritin (Santambrogio et al., 1993)
L ferritin induces iron mineralization more efficiently than H ferritin (Santambrogio et al., 1993)
L ferritin is able to withstand physical denaturation better than H ferritin because of the replacement of the ferroxidase centre of the H chain with a salt bridge in the L-chain (Santambrogio et al., 1993)

References:

Boyd, D., Vecoli, C., Belcher, D.M., Jain, S.W., & Drysdale, J.W. (1985). Structural and Functional Relationships of Human Ferritin H and L Chains Deduced from cDNA Clones. The Journal of Biological Chemistry, 260(21). 11755-11761.

Levi, S. et al. (1988). Mechanism of Ferritin Iron Uptake: Activity of the H-chain and Deletion Mapping of the Ferro-oxidase Site. The Journal of Biological Activity, 263(34). 18086-18092.

Santambrogio, P. et al. (1993). Production and Characterization of Recombinant Heteropolymers of Human Ferritin H and L Chains. The Journal of Biological Chemistry, 268(17). 12744-12748.

Sequence and Features

Assembly Compatibility: