**Abstract**

Hepcidin a 25-amino-acid and highly disulfide bonded hormone, is the central regulator of iron homeostasis. In this chapter we propose ferritin as a peptide carrier to promote the association of the hybrid hepcidin/ferritin nanoparticle with a particular cell or tissue for therapeutic or diagnostic use. Indeed, human ferritin H-chain fused directly (on its 5'end) with camel mature hepcidin was cloned into the pASK-43 plus vector and expressed using BL21 (DE3) pLys *E. coli* strain. The transformed *E.coli* produced efficiently hepcidin-ferritin construct (hepcH), consisting of 213 amino acids with a molecular weight of 24 KDa. The recovered product is a ferritin exposing hepcidin on outer surface. The hepcH monomer was characterized by immunoblotting using a monoclonal antibody specific for human ferritin and a polyclonal antibody specific for hepcidin-25. The results were also confirmed by MALDI-TOF mass spectrometry. The recombinant native human ferritin and the commercial human hepcidin-25 were used as controls in this experiment. The assembly of hepcH, as an heteropolymer molecule, was performed in presence of denatured human ferritin-H and -L chains. After cysteine oxidation of the recombinant nanoparticles, cellular binding assays were performed on mammalian cells such as mouse monocyte–macrophage cell line J774, HepG2 and COS7.

**Keywords:** camel hepcidin, chimeric nanoparticle, *engineered recombinant E. coli*, human ferritin, *protein folding*

### **1. Introduction**

The combination of chemistry, biology, and nanotechnology is expected to make significant contributions to the field of medical diagnosis and therapeutics. In this framework, the use of nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery and slow release. Protein cage architectures such as virus capsids and ferritins are versatile nanoscale platforms willing to both genetic and chemical modifications. The incorporation of multiple functionalities within these nanometer-sized protein architectures reveals their potential to serve as functional nanomaterials with applications in medical imaging and therapy. For example, RGD-4C, a cell specific targeting peptide, which binds αvβ3 integrins upregulated on tumor vasculature, was genetically incorporated on the exterior surface of a human H-chain ferritin

nanoparticle [1]. Interestingly, this modified protein cage binds specifically cancer cells in vitro. Thus, the use of ferritin cage architecture is an exciting and promising strategy to serve as a multifunctional platform for the biomimetic synthesis of magnetic nanoparticles. It can be engineered for cell-specific targeting.

Ferritin is probably the most used protein in bio-nanotechnology. This is due to its well-known structural features, high stability, capability to mineralize metals in its cavity, self-assembly and possibility to redesign its interior and exterior by protein engineering. It has been used to encapsulate molecules, for the synthesis of inorganic cores, for functional nanostructured composite material, for magnetic nanoparticles for MRI applications and for carrying various epitopes. Most published studies used the human H or L ferritin chains, which are able to self-assemble in different proportions to produce a variety of heteropolymers. This allows the possibility to adorn ferritin surface with multiple functionalities through genetic and chemical modifications to achieve desired properties for therapeutic and/ or diagnostic purposes. In particular, it can be used as a peptide carrier to target specific receptors. Unfortunately, there are no data published concerning functional biological peptides genetically fused to ferritin are missing.

In this chapter, we plan to exploit this approach by fusing hepcidin to the ferritin molecule. In fact, ferritin and hepcidin are central molecules implicated in the regulation of iron homeostasis and the fusion of the two can carry several advantages. For example, the injection of iron-loaded ferritin in a ten days wild mouse induces the expression of *BMP6* and *Hamp1* at dose-dependent manner. Such result cannot be obtained through an injection of FAC (ferric ammonium citrate) or holo-transferrin. However, the injection of uncharged ferritin has no effect on the expression of *BMP6* or *Hamp1*.

We also proposed to use ferritin to carry hepcidin, another key protein of iron metabolism. It is a small hormone peptide that control systemic iron homeostasis (ferritin is a major controller of cellular iron homeostasis). The production of chimeric ferritin complexes that expose on the surface of limited number of functional hepcidin is of interest. It is a tool that allows deep studies in relation to the mechanism of interaction between hepcidin and ferroportin and how the complex is degraded. It might indicate alternative approaches to control hepcidin activity and systemic iron homeostasis. Ferritin is composed of 24 subunits. Once defined the conditions to insert a novel function and to co-assemble different subunits in a highly stable molecule that carries are defined, it is possible to produce molecules with many more functions and it can be applied to other peptides and hormones.

The aim of this chapter is to describe an efficient strategy to fuse the full-length hepcidin to the N-terminus of ferritin-H chain (which in the assembled protein is exposed on the surface). The produced chimeric protein will be tested:


#### **2. Methods**

#### **2.1 Expression and solubilization of HepcH monomer**

Human ferritin H-chain fused directly, on its 5'end, with camel mature hepcidin was cloned into the pASK-IBA 43 plus vector (**Table 1**) and expressed using *E. coli* BL21 (DE3) pLysS. Growth of the transformed *E. coli* was done in 1 L LB medium (10 g *The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin DOI: http://dx.doi.org/10.5772/intechopen.94408*


#### **Table 1.**

*Sequences of the primes used in this study.*

Tryptone, 5 g Yeast extract, 5 g NaCl), with100μg/ml AMP, at 37°C, for 1-2 h until OD = 0,5. Expression was induced by the addition of 200 μl anhydroteracycline (1 mg/ ml), final concentration 200 μg/L, for 4 h. Cells were harvested by centrifugation at 7000 RPM for 10 min. The pallet was washed twice in Tris–HCl 20 mM pH 7,4 and sonicated, for cytoplasmic protein extraction, at the following conditions: 1 min: 30 sec, cycle5, power 72%. The sonicated pallet was centrifuged at 12000 RPM and washed twice in Tris 20 mM, 2 M Urea, 0,1% TritonX100, pH 7,4. The insoluble HepcH, precipitating with the inclusion bodies from the *E.coli* paste, was solubilized with a weight to volume ratio of 1:1 in 6 M Guanidine hydrochloride (GdnHCl) pH 4,7. The suspension was sonicated at the same conditions to homogenate the solution and incubated with stirring, for 18 h at 4°C.
