O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo. / The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.

Abstract Adenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A3 receptor (A3R) is the least explored among the four human adenosine receptor subtype members (A1, A2A, A2B and A3) and it is implicated in both neuroprotective and neurodegenerative effects. During the course of this work, the production of the recombinant human A3R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D2 receptor (D2R) was studied. A3R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A3R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A3R expression: 10µg of the monomeric A3R could be purified from one liter of insect cell culture. For successful crystallization thermostability of the A3R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A3R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D2R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D2R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D2R showed that the protein-protein interactions are calcium dependent. / Tiivistelmä Adenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A3 reseptori (A3R) on adenosiinireseptorien neljästä alatyypistä (A1, A2A, A2B ja A3) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin. Tässä työssä arvioitiin sekä ihmisen rekombinantti-A3R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D2 reseptorin (D2R) heteromerisoitumista. Rekombinantti A3 reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA3R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A3R:n tuottoon: noin 10 µg monomeerista A3R:a voitiin puhdistaa litran hyönteissoluviljelmästä. Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A3R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A3R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A3R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille. Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D2R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D2R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D2R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen.

La chromatographie mode mixte représente l’une des plus grandes évolutions de ces dernières années dans le domaine des bioséparations. Cette technique repose sur l'intervention de plusieurs types d'interactions au sein d'un seul et même support. Les résines de chromatographie mode mixte HEA, PPA et MEP HyperCel portent des groupements aliphatiques, aromatiques, thiophiliques ainsi que des groupements aminés protonables en différentes positions. Au moyen d’expériences de chromatographie, à l’aide de protéines standards aux propriétés spécifiques et de mélanges complexes, nous avons isolé ces différentes interactions. Nous avons mis en évidence l’intervention majeure d'au moins deux types d'interactions au sein de ces supports : interactions hydrophobes et électrostatiques. Nous avons pu observer le comportement des résines lors de variations de pH, de force ionique, de types de sels et de tampons ou lors de la présence d'autres composés organiques. Nous avons mis en évidence l'intervention combinée de ces types d'interactions lors des différentes phases de chromatographie. Le comportement des résines mode mixte a révélé des sélectivités particulières et dont le contrôle ciblé à l'aide de l'environnement a permis le développement de méthodes de purification efficaces et originales. Nous avons pu ainsi développer des applications telles que la purification de fragment d’anticorps (Fab’2) à partir de culture de cellules d’insectes, la capture de protéine de type MBP à partir d’extrait bactériens et la capture d’anticorps monoclonaux à partir de cellules de mammifères (CHO), et ainsi améliorer les conditions d’utilisation de la chromatographie en mode mixte. / Mixed mode chromatography is the most innovative technique for bioseparation. Mixed mode resins, as the term suggest, involves multiples types of interaction at the same time. HyperCel mixed mode resins, HEA, PPA and MEP, involve aliphatic, aromatic or thiophilic groups as well as protonable amine located in the spacer arm or as a head group. Using classical chromatographic experiments, standards proteins and complex mixtures, we highlighted the two major types of interactions involved: hydrophobic and electrostatic interactions. We specifically influenced these interactions by modifying the environment in terms of ionic strength, pH, salt types, and other compounds. The combination of these interactions during every phase of a chromatographic process has been demonstrated. Mixed mode resins thus offer unique selectivity that can be controlled by the environment. This allowed us to develop several applications from antibodies fragments capture from insect cells, to the purification of MBP-tagged proteins, through monoclonal antibody capture from CHO cells. We thus enhanced mixed mode chromatography.

A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%). / The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). Cation exchange was chosen for the last chromatography. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%).

The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.

Equistatin from the sea anemone Actinia equina contains a protein domain Eqd2 which inhibits aspartic peptidases and has not been characterized in detail. Recombinant Eqd2 was produced in the yeast expression system, and a protocol for its chromatographic purification was designed. The inhibitory specificity of Eqd2 was determined using a fluorescence inhibition assay, showing that Eqd2 is a highly selective inhibitor of cathepsin D-like and pepsin-like aspartic peptidases of family A1. Furthermore, size exclusion chromatography was used to analyze the Eqd2-peptidase complex and Eqd2 oligomerization in solution. Initial screening of crystallization conditions for Eqd2 was performed towards its structural analysis. This work provides important new information about Eqd2 as a unique type of natural inhibitors of aspartic peptidases. Its interaction mechanism can be exploited in the development of synthetic mimetics for regulation of medically important peptidases. (In Czech) Key words: peptidase inhibitors, proteolytic enzymes, activity and inhibition of enzymes, recombinant expression, protein purification, protein crystallization, equistatin

Protein Nkr-p1b is a surface receptor of cytotoxic NK cells, that mediates inhibitory signal toward the body's own cells. In this study, the ligand binding domain of the mouse protein receptor Nkr-p1b (mNkr-p1b LBD) was prepared by recombinant expression in E. coli cells. Isolated protein was subsequently used for NMR structural analysis. Prediction of protein secondary structures ratio was carried out using three different methods (CD, PSIPRED and TALOS). Results correlate well with the structure of CTLD domain, that plays a key role in ligand binding and thus to function of Nkr-p1b receptor. We managed to prepare this protein in a form suitable for NMR experiments. Based on the data obtained by NMR spectra analysis, a preliminary model of the mNkr-p1b LBD protein structure was created. However, for more precise learning of the 3D structure accurate positions of individual atoms need to be determined by other NMR spectra evaluation in the next phase. Explaining the structure of the ligand binding domain of mNkr-p1b protein could help to better understand the complex mechanism of activation of NK cell cytotoxic activity, thereby contributing to its controlled use as a therapeutic against some viral and tumor diseases.

Lactobacillus kunkeei is the most common and dominant bacterium in the honey stomach of honeybees. L. kunkeei has been isolated from honeybees all over the world. Genome sequencing has identified 5 genes for exceptionally large proteins in the genome of L. kunkeei. These proteins do not show any similarity to sequences of proteins with a known structure. These giant proteins all have a conserved region of 60 amino acids in their C-terminus. This conservation led to the hypothesis that the C-terminal domains of the giant proteins are important for their function with possibly a role in the attachment to the cell wall. In this study, a total of eight different constructs were made for two of these giant proteins. The boundaries for the constructs were determined based on bioinformatic predictions. The eight constructs all have different start positions and all end at the very C-terminal end of the protein. These constructs were cloned into an expression vector. One of the full-length giant protein was cloned into an expression vector as well.  The C-terminal constructs and the full-length proteins were recombinantly produced in Escherichia coli. Expression of six C-terminal constructs was observed and an attempt was made to purify two of the C-terminal constructs. Expression of the full-length giant protein was observed as well and purification was attempted. Neither the C-terminal constructs nor the full-length giant protein could be purified at full length. The results for the C-terminal constructs show that no folded C-terminal domain has been found for the giant proteins. A purified protein construct of the N-terminal of one of the giant proteins was available. This protein was analyzed using biophysical techniques. Circular dichroism was used to test the thermal stability. The construct did not refold after being thermally denatured. Circular dichroism measurements indicated that the N-terminal construct is composed of a mixture of α-helices and ß-sheets. Small-angle X-ray scattering data indicated that the N-terminal construct had an elongated shape with knot-like parts. Protein crystals have been obtained for the N-terminal construct and these will be analyzed using X-ray diffraction.

Time-resolved cryo-EM has already provided ground-breaking discoveries in various fields, including structural biology, biochemistry, and drug development. Compared to traditional structural biology methods where mostly stabilized conformations are reconstructed, the main advantage of time-resolved cryo-EM is its ability to capture dynamic processes in biological samples at near-atomic resolution, which allows for studying biological structures as they change and interact in real-time. In this project, I focused on the expression and purification of the individual proteins of two dynamic molecular complexes – Staphylococcus aureus (S. aureus) DNA gyrase and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication-transcription complex – and attempted to assemble them into their functional forms for cryo-EM imaging.  Both of these complexes are interesting drug targets as they play an essential role in nucleic acid replication. The function of DNA gyrase is to modulate DNA supercoiling, facilitate DNA replication, and resolve intertwined DNA molecules. The replication-transcription complex of SARS-CoV-2 comprises, among other proteins, the RNA-dependent RNA polymerase, which, together with non-structural proteins 7 and 8, is responsible for the replication of the viral genome. There are still many questions about the underlying mechanisms of these key processes, and time-resolved cryo-EM studies will provide valuable information to advance our understanding of them. Here I present expression and purification protocols for S. aureus DNA gyrase subunits A and B and SARS-CoV-2 non-structural proteins 7, 8 and 12. DNA gyrase subunits A and B were expressed in Escherichia coli (E. coli) and purified in several steps, including affinity chromatography (His-Trap), ion exchange chromatography (IEX) and size exclusion chromatography (SEC). Despite many challenges with gyrase A precipitation, I obtained enough of both subunits for the intended cryo-EM. Different strategies to assemble them into a functional tetramer were tested but did not result in the expected outcome. The gained knowledge about the behaviour of the subunits in solution will serve as a basis for further optimization of the protocols before the assembly of the complex can be attempted again. Non-structural proteins 7 and 8 were expressed in E. coli as a polyprotein and successfully purified using His-Trap and SEC. I obtained a great amount of the polyprotein and established a protocol for its cleavage. Nsp12 was expressed using the baculovirus-insect cell expression system. The immunofluorescence assay data showed that the tested lipofection protocol works, and nsp12 is being produced in sufficient quantities. This result provides a solid base for further experiments to establish a purification method and assemble the nsp12-nsp7-nsp8 complex for cryo-EM imaging.

Machupovirus är ett virus som kan orsaka hemorragisk feber hos människor. Efter utvärdering av bindning mellan designade proteiner och virala glykoproteiner skulle proteinerna potentiellt kunna användas vid framtagning av ett proteinbasserat läkemedel mot hemorragisk feber. Syftet med studien var att efter riktad evolution och framrening av optimerade varianter av proteinet AP01 undersöka inbindningen till virala glykoproteiner mellan designade AP01 proteiner och transferrinreceptorn med hjälp av flödescytometrisk undersökning. Den fysiologiska nivån av järn i kroppen upprätthålls av transferrin (Tf) och transferrinreceptorn (TfR), ett transmembranprotein bestående av tre domäner. TfR apikala domän används av glykoprotein 1 (MGP1) och Plasmodium vivax för att ta sig in i celler genom receptormedierad endocytos. Med rekombinant genteknik kan rekombinanta plasmider skapas där en gen av intresse ligeras in i en plasmid med hjälp av DNA-ligas. I studien skapades rekombinanta plasmider pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 och S3.6 som transformerades till E. coli. Erhållna resultat från sekvensering visade att samtliga sex AP01-gener hade ligerats i vektorn men sekvensering av rekombinanta plasmider visade att endast pET29b+/AP01 S2.1, S2.2, S2.3 och S3.6 hade nukleotidsekvens utan mutationer. Proteinuttryck inducerades innan proteiner renades fram med immobilized metal ion affinity chromatography (IMAC). Den uppskattade molekylvikten hos de framrenade proteinerna var 18 kDa som bestämdes med sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) vilket överrenstämde med den teoretiska molekylvikten. Flödescytometri användes för att undersöka inbindningsförmågan mellan de uttryckta proteinerna och glykoprotein 1 (MGP1). Interaktionsbindningen mellan de designade proteinerna och MGP1 är bättre än interaktionen mellan originalgen AP01 och MGP1. De designade proteinerna visar på en svag effekt i den utförda ”competition assay” som gjorts vilket kan förklaras med en ej optimal struktur hos de designade proteinerna eller närvaro av BSA. / Machupovirus is a virus that can cause hemorragic fever in humans. After evaluating the binding between designed proteins and viral glycoproteins, the proteins could potentially be used in the development of a protein-based drug for hemorrhagic fever. The aim of the study was to investigate the binding to viral glycoproteins between designed AP01 proteins and the transferrin receptor after directed evolution and purification of optimized variants of the AP01 protein by means of flow cytometric examination. The physiological level of iron in the body is maintained by transferrin (Tf) and the transferrin receptor (TfR), a transmembrane protein consisting of three domains. The apical domain of TfR is used by glycoprotein 1 (MGP1) and Plasmodium vivax to enter cells through receptor mediated endocytosis. With recombinant DNA technology, recombinant plasmids can be created where a gene of interest is ligated into a plasmid using DNA ligase. In this study, recombinant plasmids pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 and S3.6 were created and transformed into E. coli. Sequencing results showed that all six AP01 genes had been ligated into the vector but sequencing of recombinant plasmids showed that only endast pET29b+/AP01 S2.1, S2.2, S2.3 and S3.6 had nucleotid sequence without mutations. Protein expression was induced before proteins were purified by immobilized metal ion affinity chromatography (IMAC). The estimated molecular weight of the purified proteins was 18 kDa as determined by sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) which was consistent with the theoretical molecular weight. Flow cytometry was used to examine the binding ability between the expressed proteins and glycoprotein 1 (MGP1). The interaction binding between the designed proteins and MGP1 is better than the interaction between the original gene AP01 and MGP1. The designed proteins show a weak effect in the “competition assay” preformed, wich can be explained by a non-optimal structure how the designed proteins or the presence of BSA.