ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...

La synthèse de protéines recombinantes chez les microorganismes est d'un intérêt majeur pour la production de produits biopharmaceutiques, thérapeutiques et enzymatiques industriels. Cependant, la surproduction de protéines a un effet néfaste sur la physiologie cellulaire. Les ressources cellulaires (métabolites, énergie, machinerie moléculaire, espace cytosolique, etc.) sont en effet partagées entre les protéines de l'hôte et la protéine "gratuite". Cette surcharge non naturelle entraîne une croissance plus lente et des rendements en protéines plus faibles, un phénomène connu sous le nom de "burden". Dans mon projet de doctorat, il s'agissait (1) de déchiffrer les conséquences de la surproduction de protéines gratuites sur la physiologie cellulaire, (2) d'identifier le type de ressources limitantes, et (3) de surmonter cette limitation pour améliorer la production de protéines. Afin de déchiffrer les conséquences de la surproduction de protéines (1), nous avons analysé le taux de croissance, la production de protéines d'intérêt et le protéome de souches de Bacillus subtilis surproduisant divers niveaux de protéines rapportrices. Les protéines rapportrices ont été choisies de manière à être facilement quantifiables par fluorescence et par des tests d'activité (i.e. GFP, mKate2, LacZ, etc.). Pour obtenir les différents niveaux d'expression, nous avons construit des séquences synthétiques par assemblage de promoteurs constitutifs et inductibles et de régions d'initiation de traduction (TIR, RBS) variés. Nous avons ainsi montré que plus la quantité (et la taille) de la protéine produite était élevée, plus les taux de croissance étaient faibles et plus la taille des cellules était élevée. Par exemple, le taux de croissance a diminué de plus de 20 % lorsque la GFP était surproduite à plus de 5 % de la quantité totale de protéines solubles, selon des quantifications biochimiques et de fluorescence. Pour identifier le type de ressources limitantes (2), nous avons effectué une quantification relative des protéines sur les souches surproductrices de GFP et montré que certaines protéines non essentielles étaient moins abondantes dans ces souches. Nous avons ensuite dégradé spécifiquement les protéines rapportrices à l'aide d'un outil de biologie de synthèse précédemment mis au point pour B. subtilis, afin que les acides aminés puissent être recyclés dans le pool de ressources cellulaires. Avec une dégradation de 50-60% de GFP et mKate2, nous avons observé une restauration de 50% du taux de croissance. Ces résultats suggèrent que la quantité d'acides aminés (et par conséquent leur utilisation dans la synthèse des protéines) est le principal type de ressources limitantes. Pour améliorer la production de protéines (3), nous avons cherché à développer un système synthétique de recyclage des acides aminés basé sur le système de dégradation mentionné ci-dessus en surproduisant les protéases d'E. coli et B. subtilis (ClpXP) avec une protéine adaptatrice (SspB) d'E. coli. Cet outil pourrait permettre de dégrader spécifiquement des protéines non essentielles pour économiser des ressources cellulaires. Nous avons montré que la surproduction de ClpXP ou de SspB/ClpXP était suffisante pour permettre une dégradation complète des protéines produites à des niveaux bas et intermédiaires, et jusqu'à 50% des protéines fortement produites. Comme ClpXP est une protéase impliquée dans la réponse au stress, nous avons cherché à savoir si la surproduction de ClpXP pouvait avoir des conséquences négatives sur la physiologie cellulaire. Une quantification relative des protéines sur une souche surproductrice de ClpXP a montré que la surproduction de ClpXP provoque une réorganisation globale du protéome sans toutefois affecter le taux de croissance de la cellule. / Recombinant protein production in microorganisms is of great interest for the production of biopharmaceuticals, therapeutics and industrial enzymes. However, recombinant protein production has always shown a harmful effect on the microorganism cell physiology when excessively produced. Cell resources (i.e. metabolites, energy, molecular machinery, cytosolic space, etc.) are used to produce the host's proteins and the overproduced gratuitous protein. As a result, this unnatural extra load typically leads to slower growth and lower protein yields, a phenomenon known as ʻburdenʼ. This burden comes from the fact that the recombinant protein has no benefit for the microorganism, and that it only uses cell resources at the expense of the production of the endogenous essential proteins. In my PhD project, the issues were (1) to decipher the consequences of gratuitous protein overproduction on the cell physiology, (2) to identify the limiting type of resources, and (3) to overcome this limitation to improve protein production. To address the first issue (1), we analyzed growth rates, production of several proteins of interest, and genome-wide proteomes of Bacillus subtilis strains overproducing various levels of reporter proteins. The reporter proteins were chosen so that they were easily quantifiable by fluorescence and β-galactosidase activity assays (i.e. GFP, mKate2, LacZ, etc.). To obtain the various levels of expression, we built synthetic sequences made of the assembly of various constitutive and inducible promoters and translation initiation regions (TIR, RBS). Hence, we showed that higher was the amount (and size) of the protein produced, lower were the rates of growth and higher were the cell sizes. For instance, the growth rate decreased down by over 20% when GFP was overproduced above 5% of the total soluble protein amount according to both biochemical and fluorescence assays. To further identify the limiting type of resources (2), we performed a relative protein quantification on the strains overproducing GFP at different levels. Hence, we showed that some non-essential proteins were less abundant in the strains overproducing GFP. We next targeted the reporter proteins for degradation using a synthetic tool previously engineered in B. subtilis, so that amino acids can be recycled back to the pool of cell resources. Degrading the reporter gratuitous protein should also relieve the constraint on the cytosolic density by liberating intracellular space. With a degradation of 50-60% of GFP and mKate2, we observed a 50% restoration of the growth rate. This result together with the proteome analysis suggested that the amount of amino acids (and consequently their utilization in protein synthesis) was the main limiting type of resources. To overcome this limitation and improve protein production (3), we aimed at exploring a synthetic, amino acid recycling system based on the above mentioned degradation system. We decided to improve the targeted degradation system by overproducing the E. coli and B. subtilis ClpXP proteases together with an E. coli adaptor protein SspB. This tool may allow to target proteins for degradation in order to save resources and improve the production of a protein of interest. We showed that the overproduction of either ClpXP or SspB/ ClpXP were sufficient to allow a complete degradation of the proteins produced low and intermediate levels, and up to 50% of degradation of the proteins highly produced. As ClpXP is a protease involved in stress responses, we aimed to know whether the overproduction of ClpXP may have negative consequences on the cell physiology. We therefore performed relative protein quantification on a strain overproducing ClpXP. The results showed that ClpXP overproduction causes a global reorganisation on the proteome without affecting the growth rate of the cell.

Proteins containing Kunitz domain are mostly inhibitors of serine proteases. Their general characteristic is the presence of three disulfide bonds and small sizes around 6-10 kDa, although sometimes they consist of several Kunitz domains or they are part of more complex proteins. Their function is usually related to the regulation of physiological and proteolytic processes, but also to an interaction with pathogens or other defense mechanisms, such as being part of the sea anemone mucus or the venom of snakes and other invertebrates. We focused on Kunitz proteins in Eudiplozoon nipponicum, a helminth of the class Monogenea parasiting on gills of common carp (Cyprinus carpio). In the transcriptome of this parasite, several sequences with Kunitz domain have been identified based on similarities with the one already described Kunitz protein, EnKT1, suggesting that this parasite, like other bloodfeeding parasites, uses a whole set of these serine protease inhibitors with other specific functions. Several sequences with the Kunitz domain found in the transcriptome were verified by PCR and optionally supplemented by RACE-PCR. One protein, called EnKC1, was subsequently produced by recombinant expression in E. coli cells of SHuffleTM and Rosetta Gami B strains. Recombinant protein with the Kunitz domain...

The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in Escherichia coli. The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein. The influence of feed rate on solubility and proteolysis was investigated using the lacUV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same. The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter PmalK. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm. Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. E.coli strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity. / QC 20101112

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.

L-asparaginase (EC 3.5.1.1), a therapeutic enzyme used in the treatment of childhood acute lymphoblastic leukemia (ALL). Hence, the goal of this work is study the expression and evaluation of hydrolysis activity of native sequence (X12746) encoding for L-asparaginase from Erwinia chrysanthemi NCPBB1125 in the popular expression system Pichia pastoris. The sequence of asn encoded for mature protein was expressed in P. pastoris SMD1168 and X33. SDS-PAGE analysis showed recombinant L-asparaginase was secreted efficiently. Stable and high hydrolysis activity of extracellular L-asparaginase in P. pastoris SMD1168 making it a potential candidate to produce recombinant protein. After purification, a specific band whose appearance approximately 45 kDa indicating the glycosylated protein with specific activity by 6.251 Umg-1 and about 3 folds purifications. / L-asparaginase (EC 3.5.1.1), một loại enzyme được sử dụng trong điều trị bệng ung thư bạch cầu mãn tính ở trẻ em. Mục tiêu của nghiên cứu này là biểu hiện và đánh giá hoạt tính thủy phân của L-asparaginase mã hóa bởi đoạn gene (X12746) tương ứng từ Erwinia chrysanthemi NCPBB1125 được biểu hiện trong nấm men Pichia pastoris. Gene đã được cắt signal peptide và biểu hiện trong P. pastoris SMD1168 and X33. Qua phân tích kết quả điện di SDS-PAGE của môi trường sau lên men, L-asparaginase tái tổ hợp được tìm thấy trong dịch ngoại bào của P. pastoris. Với khả năng sản xuất protein có hoạt tính cao hơn so với chủng P. pastoris X33, SMD1168 được lựa chọn để biểu hiện L-asparaginase tái tổ hợp. Sau khi tinh sạch, sự xuất hiện của một băng có kích khối lượng phân tử xấp xỉ 45 kDa trên điện di SDS-PAGE cho thấy protein tái tổ hợp đã bị glycosyl hóa với hoạt tính riêng 6.251 Umg-1 và đạt độ sạch 3.471 lần.

<p>The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system.</p><p>CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction.</p><p>Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures.</p>

English abstract Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane bound glycoprotein that belongs to the metallopeptidase M28 family. Two physiological substrates were found for GCPII. The first one, N-acetyl-aspartylglutamate (NAAG), serves as a neurotransmiter in the brain and GCPII hydrolyzes it to yield free glutamate in the synaptic cleft. Excess glutamate might be cytotoxic and eventually lead to excitoxic nerve cells death. Inhibition of NAAG hydrolyzing activity has been shown to be neuroprotective. Therefore, GCPII inhibition was suggested as a therapeutic target in treatment of neurological disorders where excess glutamate is involved. The second substrate, polyglutamyl folate, is a precursor of folic acid which is required for cell growth and development. GCPII cleaves off glutamate from dietary folates and thus facilitates their absorption in small intestine. Although GCPII biological relevance is known only in the brain and the small intestine, its role in the prostate is also important. GCPII has been described as a prostate cancer marker as it is expressed on the membrane of prostate cancer cells. Since GCPII is type II transmembrane protein, it is enzymatically active and undergoes internalization, it has been suggested as a promising tool for specific anticancer-drug...

Toxoplasma gondii é um protozoário coccídio intracelular obrigatório conhecido por sua habilidade em parasitar uma ampla gama de espécies hospedeiras. A região apical do parasito é rica em organelas que, em função dos produtos liberados, estão envolvidas no processo de adesão e invasão da célula hospedeira. As proteínas liberadas por micronemas (MICs), solúveis e transmembrana, possuem domínios adesivos essenciais para a virulência do parasita. Algumas dessas proteínas são encontradas associadas entre si na superfície do taquizoíta, formando complexos como TgMIC1/MIC4/MIC6 e TgMIC3/MIC8. Em estudos anteriores demonstramos a que o subcomplexo TgMIC1/MIC4 (Fração LAC+) liga-se à lactose e estimula macrófagos a secretar IL-12. Verificamos, utilizando células HEK293 transfectadas, que um dos principais mecanismos responsáveis pela produção de IL-12 decorria do reconhecimento de N-glicanos do ectodomínio de TLR2 pelo domínio de reconhecimento de carboidrato de TgMIC1. O objetivo do presente estudo foi o de investigar a capacidade de TgMIC1 e TgMIC4 de ativar macrófagos murinos e qual o papel desempenhado por TLR2 e/ou TLR4 no desencadeamento dessa ativação. Mostramos que macrófagos derivados de medula óssea de camundongos C57Bl/6, estimulados com TgMIC1 e TgMIC4, utilizadas isoladamente ou em combinação, secretam altos níveis de citocinas pró-inflamatórias, como TNF-?, IL-6, IL-12 e IL-1?, produzem altos níveis de óxido nítrico, e têm aumentadas suas capacidades migratória e fagocítica. Os ensaios que utilizaram macrófagos de camundongos C57Bl/6 nocauteados revelaram que a ausência de expressão de TLR2 ou de TLR4 prejudicou os efeitos ativadores exercidos por TgMIC1 e TgMIC4. Macrófagos TLR2-/- tiveram as manifestações de ativação celular significantemente reduzidas em relação aos macrófagos selvagens. Por outro lado, esses efeitos foram mais afetados pela ausência de TLR4, uma vez que as respostas obtidas frente ao estímulo com TgMIC1 ou TgMIC4 eram similares às verificadas em células não estimuladas (controle negativo). Concluímos que TgMIC1 e TgMIC4 interagem com os receptores do tipo toll 2 e 4 expressos por macrófagos, levando à ativação celular manifesta por alta produção de citocinas e outros mediadores inflamatórios, e aumento das capacidades migratória e fagocítica. A interação com TLR4 é preponderante em relação à estabelecida com TLR2 no desencadeamento de ativação celular / Toxoplasma gondii is an obligate intracellular coccidian protozoan known for its ability to parasitize a wide range of host species. The parasite\'s apical region is rich in organelles that, because of the products it releases, are involved in the processes of adhesion and invasion of the host cell. The soluble and transmembrane proteins released by the micronemes (MICs), have adhesive domains that are essential for the parasite virulence. Some of these proteins can be found associated with each other on the taquizoite surface, as it is the case of TgMIC1/MIC4/MIC6 or TgMIC3/TgMIC8 complexes. Our group has demonstrated in previous studies that the TgMIC1/TgMIC4 subcomplex (LAC+ fraction) binds to lactose and stimulates macrophages to release IL-12. Using HEK293 transfected cells, we showed that one of the main mechanisms leading to IL-12 release by macrophages, was the recognition of N-glycans of the TLR2 ectodomain by the carbohydrate recognition domain of TgMIC1. Thus, the objective of this study was to address whether TgMIC1 and TgMIC4 activate murine macrophages and what is the role of TLR2 and TLR4 in macrophage activation. Our results show that the stimulation of C57BL/6 mice bone marrow derived macrophages with TgMIC1 and TgMIC4, alone or in combination, induce the release of high levels of proinflammatory cytokines such as TNF-?, IL-6, IL-12 and IL-1?, and also nitric oxide; and increases phagocytic activity and cell migration activity. The assays using knockout C57Bl/6 macrophages showed that the absence of TLR2 or TLR4 expression impaired the activating effects of TgMIC1 e TgMIC4. TLR2-/- macrophages presented significantly reduced cell activation manifestations compared to WT macrophages. On the other hand, these effects were more affected in the absence of TLR4, once the responses obtained with TgMIC1 or TgMIC4 stimulation were similar to those observed in non stimulated cells (negative control). We conclude that TgMIC1 and TgMIC4 interact with the receptors Toll like 2 and 4 expressed in macrophages, leading to cell activation, resulting in high cytokine and inflammatory mediators production, and increased migratory and phagocytic capacity. The interaction with TLR4 is predominant over that established with TLR2 in the triggering of cell activation