Academic literature on the topic 'Protein nanocages'

Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

Dps (DNA-binding protein from starved cells) is well known for the structural protection of bacterial DNA by the formation of highly ordered intracellular assemblies under stress conditions. Moreover, this ferritin-like protein can perform fast oxidation of ferrous ions and subsequently accumulate clusters of ferric ions in its nanocages, thus providing the bacterium with physical and chemical protection. Here, cryo-electron microscopy was used to study the accumulation of iron ions in the nanocage of a Dps protein from Escherichia coli. We demonstrate that Fe2+ concentration in the solution and incubation time have an insignificant effect on the volume and the morphology of iron minerals formed in Dps nanocages. However, an increase in the Fe2+ level leads to an increase in the proportion of larger clusters and the clusters themselves are composed of discrete ~1–1.5 nm subunits.

The SARS-CoV-2 pandemic has created a global public crisis and heavily affected personal lives, healthcare systems, and global economies. Virus variants are continuously emerging, and, thus, the pandemic has been ongoing for over two years. Vaccines were rapidly developed based on the original SARS-CoV-2 (Wuhan-Hu-1) to build immunity against the coronavirus disease. However, they had a very low effect on the virus’ variants due to their low cross-reactivity. In this study, a multivalent SARS-CoV-2 vaccine was developed using ferritin nanocages, which display the spike protein from the Wuhan-Hu-1, B.1.351, or B.1.429 SARS-CoV-2 on their surfaces. We show that the mixture of three SARS-CoV-2 spike-protein-displaying nanocages elicits CD4+ and CD8+ T cells and B-cell immunity successfully in vivo. Furthermore, they generate a more consistent antibody response against the B.1.351 and B.1.429 variants than a monovalent vaccine. This leads us to believe that the proposed ferritin-nanocage-based multivalent vaccine platform will provide strong protection against emerging SARS-CoV-2 variants of concern (VOCs).

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein–protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.

The delivery of therapeutic proteins is one of the greatest challenges in the treatment of human diseases. In this frame, ferritins occupy a very special place. Thanks to their hollow spherical structure, they are used as modular nanocages for the delivery of anticancer drugs. More recently, the possibility of encapsulating even small proteins with enzymatic or cytotoxic activity is emerging. Among all ferritins, particular interest is paid to the Archaeoglobus fulgidus one, due to its peculiar ability to associate/dissociate in physiological conditions. This protein has also been engineered to allow recognition of human receptors and used in vitro for the delivery of cytotoxic proteins with extremely promising results.

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.
As nanogaiolas proteicas apresentam uma estrutura quase esférica e são capazes de incorporar enzimas ativas e/ou intermediários ou produtos tóxicos na sua gaiola oca, à parte do resto da célula. Nesta tese, a Dps de Marinobacter hydrocarbonoclasticus (proteína 12-mer) foi encapsulada no interior da EncA de Myxococcus xanthus (composta por 180 subunidades), criando um sistema gaiola-dentro-de-gaiola (EncA:DpsT). Para atingir este objetivo, uma sequência sinal necessária para o encapsulamento foi inserida no C-terminal da Dps (DpsT) levando à incorporação de ~ 8 moléculas de DpsT dentro de cada encapsulina. Técnicas bioquímicas e espectroscópicas confirmaram a estrutura global nativa das proteínas expressas em Escherichia coli, exibindo pequenas diferenças quando comparadas com as suas formas nativas individuais. Durante este trabalho, as proteínas Dps WT, DpsT, EncA e EncA:DpsT foram avaliadas com base na sua termoestabilidade, resistência proteolítica e capacidade de ligação ao ADN. Embora o carácter positivo da sequência sinal inserida na Dps tenha diminuído a termoestabilidade da mini-ferritina em comparação com a sua forma nativa, a afinidade de ligação ao ADN aumentou 500 vezes. A EncA e o complexo EncA:DpsT mostraram também ser capazes de ligar ADN plasmídeo superenrolado. Além disso, o invólucro da encapsulina forneceu um escudo físico para a proteína DpsT encapsulada, como demonstrado pelos ensaios de proteólise com Proteinase K. A capacidade destas proteínas para incorporação ferro foi também avaliada, utilizando H2O2 como agente oxidante, de modo a estimar a sua capacidade máxima de armazenamento de ferro. Enquanto a Dps WT e a DpsT incorporaram a mesma capacidade de ferro (~ 1.000 átomos de ferro), o complexo EncA:DpsT revelou ser capaz de incorporar o dobro da quantidade de ferro relativamente à EncA sozinha (12.000 vs 6.000 átomos de ferro). Além disso, para estudar a cinética de oxidação e mineralização do ferro, foram realizados ensaios com O2 como oxidante e o cobre foi utilizado como catalisador putativo no processo de ferroxidação. O perfil cinético do complexo EncA:DpsT foi semelhante ao da EncA e da DpsT. No entanto, o encapsulamento reduziu a atividade catalítica da DpsT, na presença de cobre. Embora tanto a EncA vazia como a complexada tenham reduzido a quantidade total de ferro incorporado com sucesso na presença de cobre, o encapsulamento da DpsT resultou na incorporação de uma maior quantidade de ferro pelo sistema, em comparação com a EncA vazia. O presente trabalho relata, pela primeira vez, a produção de um sistema de gaiola-dentro-de-gaiola através de engenharia genética da proteína Dps. Assim, o trabalho desenvolvido nesta tese fornece uma visão da cooperação entre estas duas proteínas abrindo, ao mesmo tempo, novas aplicações para os sistemas de encapsulinas.

碩士
國立臺灣大學
醫學工程學研究所
103
Currently clinical treatment for colorectal cancer, in addition to surgical, adjuvant chemotherapy can significantly reduce the chances of recurrence of colorectal cancer patients. Platinum drugs mechanism of action is to block DNA synthesis, the first generation of cisplatin although have good treatment, but because of its side effects induce patient discomfort, so researchers have developed current clinical use of third generation platinum drugs oxaliplatin (Oxa). Although oxaliplatin side effects compared to cisplatin is not so high, but the side effects of nephrotoxicity and neurotoxicity affect the patient''s quality of life, so we began to combine different pharmaceutical carriers, hope with nanocarrier can enhance the efficacy and reduce side effects. Dichloro(1,2-diamino-cyclohexane)platinum(II) (DACH - Pt) is oxaliplatin active form, so in this study, we use the DACH - Pt as carried drugs. Protein carrier is a novel drug carrier, in my study I choose apoferritin (Apo), is a spherical iron storage protein composed of 24 subunits. Apo protein self-assembles naturally into a hollow nanocage with an outer diameter of 12 nm and an interior cavity 8 nm in diameter, this may lead to a longer circulation half-life and a better tumor accumulation rate, and it is a protein present in animal body, so the carrier has a high biocompatibility and biodegradable. Recently, it was reported that apoferritin binds to human cells via interacting with the transferrin receptor 1 (TfR1, CD71), and under normal circumstances the cells in order to maintain the balance of intracellular iron concentration so regard to a certain degree of expression TFR1. It is well known that TfR1 is highly expressed on human colorectal cancer cells and has long been used as a targeting marker for tumor diagnosis and therapy. The Apo nanocages can be collapse in an acidic environment (pH = 2) into subunits, and the process is reversible. When the pH is turned back to neutral, the Apo subunits will be reconstituted into a hallow structure, and almost in an intact appearance, so we can use this characteristic to achieve effectively loading into the cavity, such as pH dependent disassembly and reassembly can be exercise of construct Apo.