Ferritins are ubiquitous proteins that serve the dual-function of iron reservoir and sequestering the Fe(II) toxicity. The function of ferritins totally depends on the characteristic spherical structure with a di-iron centre performing the iron oxidation and a hallow cavity enclosing the iron minerals in a bioavailable form. I have characterised the structure, assembly and function of a new member of ferritin superfamily that is natively enclosed within an encapsulin shell. Encapsulin proteins are structurally-related to a virus capsid and form 60-meric or 180-meric icosahedrons. I show that this encapsulin associated ferritin-like protein (EncFtn) possesses two main alpha helices, which assemble in a metal-dependent manner to form a ferroxidase centre at a dimer interface. EncFtn adopts an annular decamer structure in contrast to the 24-meric classical ferritins or 12-meric mini-ferritin (DPS). The resemblance of the dimeric EncFtn and monomeric classical ferritins suggests that it is likely that classical ferritin evolves from EncFtn because of the gene duplication. EncFtn is a catalytically active ferroxidase but with only a limited iron binding ability due to its open structure. The encapsulin itself is not able to oxidise Fe(II), but is able to store about 2200 iron ions. I have demonstrated that the EncFtn must be housed in the encapsulin to achieve a maximum loading of approximately 4200 iron ions. The encapsulin nanocompartments are widely-distributed in both eubacteria and archaeon with distinct life styles and represent a distinct class of iron storage system, where iron oxidation and mineralisation are distributed between two proteins.

L’ingénierie protéique servant autrefois à comprendre les relations structures-fonctions des protéines connait un tournant majeur depuis plusieurs années. L’ingénierie protéique évolue pour créer des nouvelles fonctions protéiques : c’est la naissance de l’ingénierie protéique moderne. L’objectif de ma thèse a consisté à mettre en place et caractériser deux approches indépendantes d’ingénierie protéique dans le domaine du vaccin et du diagnostic. Le premier projet consistait à générer des ligands protéiques à partir d‘échafaudages moléculaires (des alternatifs aux anticorps) en couplant le ribosome display au NGS et en développant des outils d’analyses bio-informatiques. Des sélections contre des cibles protéiques d’origine bactérienne et virale ont conduit à l’identification de ligands Affibodies affins (µM au nM). Leur caractérisation a validé leur potentiel comme outil de recherche et de réactif diagnostique. Ces études ont permis de valider la plateforme de génération des ligands mise en place, en augmentant l’exploration de l’espace de diversité des interactions des ligands. Le second projet portait sur le développement d’une plateforme de présentation et de vectorisation à partir de particules d’encapsuline. Elles ont été génétiquement modifiées pour présenter de manière répétée à leur surface l’ectodomaine de la protéine de matrice M2 (M2e) du virus Influenza A H1N1 tout en encapsulant une protéine hétérologue : l’eGFP. Les nanoparticules modifiées sont correctement formées et encapsulent l’eGFP. Des souris immunisées par ces particules induisent une réponse anticorps spécifique contre l’épitope M2e et l’eGFP. L’utilisation de ces nanoparticules comme plateforme vaccinale de présentation et de vectorisation est prometteuse et ouvre la voie pour d’autres applications en biotechnologie / In the past, protein engineering used to understand function and structure relationship. But since few years, protein engineering was used to create new protein functions: modern protein engineering was born. The aim of my thesis was to set up and characterize two approaches of protein engineering in diagnostic and vaccine field. The first project was to generate artificial binder using protein scaffolds as an alternative to antibodies by coupling ribosome display (RD) to NGS and developing bio-informatics tools. Screening and selection against bacterial and viral targets have led to affibody binder’s identification with an affinity range from µM to nM. Their characterization has validated their potential as research tools and protein reagents for diagnostic assay. Coupling ribosome display to high throughput sequencing as means to directly identify selected binder coding sequences, enormously enhance binder discovery depth. The second project was to generate an innovative nanocarrier based on encapsulin nanoparticle, for customized peptide display and cargo protein vectorization. Encapsulin particles from T.maritima were genetically modified for simultaneous display of the matrix protein 2 ectodomain of the influenza H1N1 A virus and heterologous protein eGFP packaging. Genetically engineered encapsulin nanoparticles were well-formed and abled to efficiently load eGFP. Immunogenicity studies revealed antibody responses against both the surface epitope and the loaded cargo protein. Taken together, this display system is a versatile tool for rational vaccine design and paves the way for new applications in the research fields of vaccine, antimicrobial research and other biotechnological applications