Recombinant preparation of TEAD transcription factor (abstract) The TEAD family transcription factors play an important role during devolopment of organisms, where their main purpose is to regulate organ size by activating expression of proteins involved in cell growth and differentiation and apoptosis inhibition. TEAD proteins activity is regulated by signalling pathways and interactions with coactivators. Disregulation of these mechanisms can lead to development of tumors, which is the reason why TEAD proteins became an interesting target for development of new anticancer drugs based on inhibiting their activity. There are several possibilities how to inhibit activity of a transcription factor including blocking its bond to DNA. To design a new drug that blocks transcription factors binding to DNA the structural basis of interaction of these two molecules has to be known first. In this thesis the DNA binding domain of human protein TEAD1 was prepared using the technique of recombinant expression in bacteria E. coli. Suitable conditions of protein production were found and the DNA binding domain of TEAD1 protein was purified so it will be possible to use it for structural analysis of its intraction with DNA.

Les patients atteints de cécité cornéenne résultant d'une maladie ou d'une blessure dans de nombreux pays ne seront probablement pas transplantés avec des cornées de donneurs humains en raison d'une grave pénurie mondiale de tissus de donneurs. Cependant, même si des cornées de donneurs étaient disponibles, les patients présentant une inflammation ou une maladie grave ne seraient pas aidés car ils courent un risque élevé de rejet des cornées de donneurs car celles-ci contiennent des cellules allogéniques. Les implants cornéens sans cellules qui ne déclenchent pas de rejet ont été développés comme alternatives à la transplantation de donneurs humains par le laboratoire Griffith, et ont montré dans un premier essai clinique chez l'homme qu'ils régénèrent de manière stable le tissu et les nerfs cornéens. Ces implants comprenaient du collagène humain recombinant, la principale protéine structurelle trouvée dans la cornée humaine. Cependant, les collagènes de pleine longueur sont difficiles et coûteux à produire et ne peuvent pas être personnalisés. Une grande variété de peptides plus courts qui imitent le collagène et d'autres molécules de la matrice extracellulaire ont été développés et testés. Cela comprend les peptides hybrides combinant le collagène et la soie (VBsilk). Le but de ma thèse est de confirmer les simulations de VBsilk d'un peptide hybride collagène-soie produit au Griffith Lab. Un autre objectif est de déterminer les conditions de production et de purification pour montrer que le peptide simulé peut être converti en un peptide réel. En bref, l'ADN codant pour une séquence de VBsilk a été cloné dans ClearColi, une souche d'E. Coli à faible endotoxine. Les bactéries ont été cultivées dans des cultures à grand volume. Le VBsilk a été extrait et purifié par FPLC. SDS-PAGE a montré que des bandes de protéines de taille appropriée étaient obtenues. Par conséquent, il est possible de produire le peptide VBsilk. / Patients with cornea blindness resulting from disease or injury in many countries are unlikely to be transplanted with human donor corneas due a worldwide severe shortage of donor tissues. However, even if donor corneas were available, patients with inflammation or severe disease would not be helped as they are at a high risk of rejecting donor corneas as these contain allogeneic cells. Cell-free corneal implants that do not trigger rejection were developed as alternatives to human donor transplantation by the Griffith lab, and shown in a first-in-human clinical trial to stably regenerate corneal tissue and nerves. These implants comprised recombinant human collagen, the main structural protein found in the human cornea. However, full-length collagens are difficult and expensive to produce, and cannot be customized. A wide variety of shorter peptides that mimic collagen and other extracellular matrix molecules have been developed and tested. This includes hybrid peptides combining collagen and silk (VBsilk). The aim of my thesis is to is to confirm simulations of VBsilk, a hybrid collagen-silk peptide that was produced in the Griffith Lab. A further aim is to determine the conditions for the production and purification to show that simulated peptide can be converted into an actual peptide. Briefly, the DNA coding for a VBsilk sequence was cloned into ClearColi, a strain of E. coli with low endotoxin. The bacteria were grown up in large volume cultures. The VBsilk was extracted and purified by FPLC. SDS-PAGE showed that appropriate-sized bands of protein were obtained. Hence, it is possible to produce VBsilk peptide.

Philosophiae Doctor - PhD / Tumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin. / 2022

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Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin cleavage site was incorporated proximal to the tag, followed by cloning in Pichia pastoris, methanol-induced expression, and cobalt-affinity chromatography for initial purification stages. Notably, the recombinant tags did not exhibit a discernible influence on Cp3GT kinetics, substrate preference, pH optima, or metal interactions, maintaining its specificity towards flavonols at the 3-OH position and favoring glucosylation of quercetin and kaempferol. Subsequent purification steps, including MonoQ anion exchange and size-exclusion chromatography, yielded Cp3GT with ≥95% homogeneity. In silico molecular models of Cp3GT and its truncated variants, Cp3GTΔ80 and Cp3GTΔ10, were constructed using D-I-TASSER and COFACTOR to assess binding interactions with quercetin and kaempferol. Results indicated minimal interference of c-myc/6x-his tags with the native Cp3GT structure. This study not only lays a foundation for impending crystallographic studies, aiming to solidify the understanding of Cp3GT's stringent 3-O flavonol specificity, but also accentuates the potential of microbial expression platforms and plant metabolic engineering in producing beneficial compounds. To this end, a thorough review of four pivotal classes of plant secondary metabolites, flavonoids, alkaloids, betalains, and glucosinolates, was conducted. This will open avenues for further research and applications in biotechnological, medical, and agricultural domains.

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Lysosomal storage diseases are genetically inherited diseases caused by the dysfunction of lysosomal enzymes. In a normal cell, lysosomal enzymes cleave specific macromolecules as they are transported to the lysosome. However, in diseased cells, these lysosomal enzymes are either absent or malfunctioning, causing macromolecular substrates to accumulate, becoming toxic to the cell. Over fifty lysosomal storage diseases have been identified, collectively occurring in one out of 7,700 live births. We investigated the lysosomal enzyme β-galactosidase (β-gal). In order to study the biochemistry and enzymology of this protein a robust expression system was needed. The GLB1 gene has been inserted into Pichia pastoris creating high protein expressing cell lines. The result of this work will yield a high expression system for β-gal, which can then be subjected to structural and biochemical studies.