Using protein nanocages for biochemical and biotechnological applications

• Autor(a) principal: Carvalho, Ana de Jesus Silva
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/130337
• topic: Dps proteins; Encapsulins; Protein encapsulation; Iron ferroxidation and mineralization; Copper as a catalyst; DNA binding and protection; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Nanocage proteins exhibit a near spherical structure and are able to incorporate active enzymes and/or harmful intermediates or products in their hollow cage, apart from the rest of the cell. In this thesis, Dps from Marinobacter hydrocarbonoclasticus (a 12-mer protein) was encapsulated inside EncA from Myxococcus xanthus (composed of 180 subunits), creating a cage-in-a-cage system (EncA:DpsT). To accomplish that, a tag required for the encapsulation was inserted at the C-terminal of Dps (DpsT) leading to the incorporation of ~ 8 DpsT molecules inside each encapsulin shell. Biochemical and spectroscopic techniques confirmed the overall native structure of the proteins expressed in Escherichia coli, exhibiting small differences while comparing to the individual native forms. During this work, Dps WT, DpsT, EncA and EncA:DpsT proteins were evaluated based on their thermostability, proteolytic resistance and DNA binding ability. Although the positive character of the tag inserted in Dps decreased the thermostability of the mini-ferritin compared to its native form, the DNA binding affinity increased 500 times. EncA and EncA:DpsT complex were also capable of binding supercoiled plasmid DNA. Moreover, the encapsulin shell provided a physical shield for the cargo DpsT, as demonstrated by proteolysis assays with Proteinase K. The uptake of iron by these proteins was also evaluated, using H2O2 as oxidant agent to estimate their maximum iron loading capacity. While Dps WT and DpsT showed to have the same iron capacity (~ 1,000 iron atoms), EncA:DpsT complex revealed twice the amount of iron incorporated by the encapsulin nanocage alone (12,000 vs 6,000 iron atoms). Additionally, to study the oxidation and mineralization kinetics, assays with O2 as oxidant were performed and copper was used as a putative catalyst in the ferroxidation process. The overall kinetic profile of the EncA:DpsT complex was similar to the EncA and DpsT ones. The presence of copper increased the kinetic rate for all proteins. Nevertheless, the encapsulation reduced the catalytic activity of DpsT when copper was used. Although both empty and cargo loaded EncA reduced the total amount of iron successfully incorporated when copper was present, the encapsulation of DpsT improved the uptake of a larger amount of iron by the EncA:DpsT system, compared to EncA alone. The present work reports, for the first time, the production of a cage-in-a-cage system by engineering a Dps protein. Thus, the work developed in this thesis provides insight into the cooperation between these two proteins, while opening new applications for the encapsulin systems.