>Magister Scientiae - MSc / The yeast Saccharomyces cerevisiae is considered a good host used for heterologous protein production due to the organism’s microbial safety, rapid growth and eukaryotic post- translational processing. As a fermentative organism, S. cerevisiae is thus not only a useful platform for the production of biopharmaceuticals and industrial enzymes, but also a promising organism for second-generation biofuel production. Substantial effort has been focused on alleviating the many bottlenecks in recombinant gene expression, as well as in the secretory pathway to enhance heterologous protein titres. It was recently shown that highly active endocytosis could decrease the overall secreted protein titre in the supernatant. In this study, we aimed to block endocytotic and vacuolar complexes to ultimately disrupt, or impair, the endocytotic and vacuolar mechanisms of proteolysis and test the effect that this would have on secreted heterologous protein titres. This was accomplished by knocking out various genes involved in endocytosis and transforming the strains with genes encoding various hydrolases including β-glucosidase (Bgl), xylanase (Xyn2), endoglucanase (Eg2) and cellobiohydrylase (Cbh1). Our study demonstrated that genetic blocking of endocytotic mechanisms as well as vacuolar complexes could theoretically improve heterologous protein secretion in S. cerevisiae. Endoglucanase (Eg2) titres displayed improvement of 26% and 30% in strains which had the RVS161 and VRP1 genes deleted and xylanase titres displayed an improvement of 71% and 143% in strains with the END3 and SSA4 gene deletions. Several of the gene knockouts tested improved Xyn2 and Eg2 titres but the effect of the different gene targets varied widely. A double knock-out strain with deletions in CLC1 and RVS161 secreted 104% more Eg2 than its parental control strain on a per dry cell weight basis, a significant synergistic improvement. Other double knock-out strains displayed additive or similar activities when compared to their controls. Cbh1 secretion could not be improved through the gene deletions tested in our study and Bgl activity could not be measured in our transformants. These results demonstrate the different relationships of various heterologous proteins with various components of the secretion machinery and may also imply how endocytosis as well as vacuolar complexes affect the level of secreted protein.

NK (natural killer) cells, with their ability to identify antigens and extraneous substances, available in the organism through various moleculary receptors, are an important component of the immune system. The NKR-P1A and NKR-P1B proteins belong to the lectin receptors of natural killer cells. Primary ligands of lectin receptors comprise terminal oligosaccharides of glycoproteins on the surface of target (e.g. tumor) cells. The interaction between carbohydrate structures on the surface of antigens and their binding partners on NK receptors is followed by triggering the effector function of NK cells against the targets. The NK cells and NK receptors findings and their interactions with ligands are greatly utilized in the treatment of cancer, viral and autoimmune diseases. Heterologous protein production in the eukaryotic organism brings a lot of advantages. Unlike the prokaryotic organism, the methylotrophic yeast Pichia pastoris has the capability of performing many posttranslational modifications resulting in production of biological active protein molecule. Usually, the P. pastoris expression system disposes of high level protein expression and is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from other eukaryotes. In this thesis, I...

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Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) είναι κατιονικοί δίαυλοι, οι οποίοι ενεργοποιούνται κατόπιν πρόσδεσης κατάλληλου νευροδιαβιβαστή και ευθύνονται για την κυτταρική επικοινωνία δια μέσου της ακετυλοχολίνης. Ο κυριάρχος τύπος νικοτινικών υποδοχέων στον εγκέφαλο των θηλαστικών με υψηλή συγγένεια για νικοτίνη, αποτελείται από α4 και β2 υπομονάδες. Ο συγκεκριμένος τύπος υποδοχέων, είναι υπεύθυνος για την εξάρτηση στην νικοτίνη και θεωρείται ότι εμπλέκεται στις ασθένειες Alzheimer και Parkinson. Για το λόγο αυτό οι συγκεκριμένοι δίαυλοι, αποτελούν έναν σημαντικό στόχο σχεδιασμού και ανάπτυξης φαρμάκων. Στην προσπάθειά μας να αποκτήσουμε υδρόφιλες περιοχές του ανθρώπινου υποδοχέα α4β2 για δομικές μελέτες ικανές να προσδένουν αγωνιστές, εκφράσαμε τα εξωκυττάρια τμήματα των συγκεκριμένων υπομονάδων στο ευκαρυωτικό σύστημα έκφρασης ζύμης Pichia pastoris. Στα αγρίου τύπου εξωκυττάρια τμήματα αλλά και στις μεταλλαγμένες μορφές τους, στις οποίες έχει αντικατασταθεί η κυστινική θηλιά με την περισσότερο υδρόφιλη αντίστοιχη περιοχή της πρωτεΐνης η οποία δεσμεύει ακετυλοχολίνη, δεν παρατηρήθηκε καμία ειδική αλληλεπίδραση με γνωστούς προσδέτες. Κρίθηκε λοιπόν αναγκαίο να εκφράσουμε τα εξωκυττάρια τμήματα διασυνδεόμενα με ένα ολιγοπεπτίδιο 24 αμινοξικών καταλοίπων (AGS)8, ως ένα μόριο. Παρατηρήθηκε λοιπόν ότι το συγκαταμερές β2-24-α4-mECD αλλά όχι το α4-24-β2-mECD είναι αρκετά υδατοδιαλυτό μόριο με πολύ καλές ιδιότητες πρόσδεσης αγωνιστών. Η ιωδινιωμένη επιβατιδίνη (125Ι-επιβατιδίνη) και η τριτιωμένη νικοτίνη ([3Η]-νικοτίνη) δεσμεύονται στο συγκαταμερές β2-24-α4-mECD με σταθερές διάσπασης (Kd) 0,38 και 19 nM αντίστοιχα, τιμές οι οποίες προσεγγίζουν τις αντίστοιχες γνωστές από τη βιβλιογραφία, που προσδίδονται σε ολόκληρο τον ανθρώπινο υποδοχέα α4β2. Επιπλέον, το πόσο ειδική είναι η δέσμευση της 125Ι-επιβατιδίνης φαίνεται και από το γεγονός ότι παρεμποδίζεται συναγωνιστικά από τους αγωνιστές νικοτίνη, κυτισίνη, ακετυλοχολίνη και καρβαμυλοχολίνη με τιμές σταθεράς αναστολής (Κi) ίσες με 20,64, 3,24, 242 και 2254 nM αντίστοιχα. Επιπλέον, επιχειρήθηκε η δημιουργία πενταμερών εξωκυττάριων τμημάτων των ανθρώπινων υπομονάδων α4 και β2. Αρχικά, πραγματοποιήθηκε συνέκφραση καθενός από τα δύο συγκαταμερή (α4-24-β2-mECD και β2-24-α4-mECD), τα οποία φέρουν χαρακτηριστική αλληλουχία έξι ιστιδινών (6xHis), με κάθε εξωκυττάριο τμήμα των μεταλλαγμένων υπομονάδων α4 ή β2 τα οποία φέρουν την χαρακτηριστική αλληλουχία του οκταπεπτιδίου FLAG. Στις περιπτώσεις της συνέκφρασης του συγκαταμερούς α4-24-β2-mECD με καθένα από τα εξωκυτταρικά και μεταλλαγμένα τμήματα των υπομονάδων α4 ή β2 (α4-mECD ή β2-mECD) δεν παρατηρήθηκε δέσμευση με 125Ι-επιβατιδίνη και συνεπώς δεν προχωρήσαμε σε περαιτέρω χαρακτηρισμό. Μόνο στην περίπτωση της συνέκφρασης του συγκαταμερούς β2-24-α4-mECD με το β2-mECD παρατηρήθηκε πρόσδεση με 125Ι-επιβατιδίνη. Επίσης, οι μελέτες δυναμικής σκέδασης φωτός εμφάνισαν μία εκτεταμένη ετερογένεια μεγέθους των τελικών προϊόντων όπως απομονώνονται μέσω της χρωματογραφίας μοριακής διήθησης, αποκλείοντας, τουλάχιστον σε αυτό το σημείο οποιαδήποτε προσπάθεια δομικής μελέτης για τα συγκεκριμένα μόρια. Κατασκευάστηκαν επίσης δύο συγκαταμερή τριμερών (β2-24-β2-24-α4-mECD και β2-24-α4-24-β2-mECD). Δυστυχώς, η ανάλυση μέσω SDS-PAGE και η αποτύπωση κατά Western αποκάλυψε την ύπαρξη ενός μείγματος, το οποίο αποτελείται από ολόκληρα τα μόρια (τριμερή) αλλά και από πολυπεπτίδια στα οποία απουσιάζουν ένα ή και δύο από τα μονομερή εξωκυττάρια τμήματα (διμερή και μονομερή αντίστοιχα). Το συγκεκριμένο αποτέλεσμα υποδηλώνει ότι το σύστημα ετερόλογης έκφρασης το οποίο χρησιμοποιούμε (P. pastoris) αν και είναι κατάλληλο για την έκφραση των λειτουργικών διμερών συγκαταμερών (β2-24-α4-mECD), είναι μάλλον ακατάλληλο για την έκφραση πιο περίπλοκων κατασκευών, πιθανότατα εξαιτίας γεγονότων ομόλογου ανασυνδιασμού των πλασμιδίων πριν ή κατά την ενσωμάτωσής τους στο γονιδίωμα της ζύμης. Ακόμα, ενδιαφέρον είναι το γεγονός ότι το απογλυκοζυλιωμένο συγκαταμερές β2-24-α4-mECD εμφανίζει τις ίδιες ιδιότητες πρόσδεσης αγωνιστών. Επιπροσθέτως, το απογλυκοζυλιωμένο συγκαταμερές β2-24-α4-mECD απομονονέται ως μονομερές, γεγονός το οποίο, σε συνδιασμό με την προηγούμενη παρατήρηση, καθιστά την συγκεκριμένη πρωτεΐνη κατάλληλη να χρησιμοποιηθεί σε δομικές μελέτες και πιθανόν για την φαρμακολογική εκτίμηση καινούργιων και ειδικών αγωνιστών για υποδοχείς α4β2. / Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels responsible for cell communication via the neurotransmitter acetylcholine. The predominant nAChR subtype in the mammalian brain with a high affinity for nicotine is composed of α4 and β2 subunits. This nAChR subtype is responsible for addiction to nicotine and is thought to be implicated in Alzheimer and Parkinson diseases and therefore presents an important target for drug design. In an effort to obtain water-soluble, ligand-binding domains of the human α4β2 nAChR appropriate for structural studies, we expressed the extracellular domains (ECDs) of these subunits in the eukaryotic expression system Pichia pastoris. The wild-type ECDs and their mutants containing the more hydrophilic Cys-loop from the snail acetylcholine-binding protein (individually expressed or coexpressed) did not demonstrate any specific interaction with ligands. We then linked the mutated ECDs with the 24 amino acid peptide (AGS)8 and observed that the β2-24-α4-mECD concatamer, but not the α4-24-β2-mECD one, exhibited very satisfactory water solubility and ligand binding properties. The 125I-epibatidine and [3H]nicotine bound to β2-24-α4-mECD with dissociation constants (Kd) of 0.38 and 19 nM, respectively, close to the published values for the intact α4β2 nAChR. In addition, 125I-epibatidine binding was blocked by nicotine, cytisine, acetylcholine, and carbamylcholine with inhibition constants (Ki) of 20.64, 3.24, 242, and 2,254 nM, respectively. In addition, we attempted to create pentameric domains of the extracellular human subunits α4 and β2. Initially, we accomplished the coexpression of the particular concatamers, α4-24-β2-mECD and β2-24-α4-mECD, bearing the six histidine tag (6xHis), with each of the mutated extracellular domains of the subunits α4 and β2 (α4-mECD and β2-mECD), bearing the characteristic octapeptide FLAG tag. The coexpression of the concatamer α4-24-β2-mECD with each of α4 or β2-mECD did not lead to binding to 125I - epivatidine and therefore we did not proceed to any further characterization. Only in the case of the coexpression of the concatamer β2-24-α4-mECD with the β2-mECD we observed binding to 125I - epivatidine. Aditionally, the dynamic light scattering studies demonstrated a widespread size heterogeneity of the final product as isolated by gel filtration chromatography, blocking, at least at this point, any further attempt for structural studies of the specific molecules. We then constructed two trimeric concatamers (β2-24-β2-24-α4-mECD and β2-24-α4-24-β2-mECD) . Unfortunately, the analysis by SDS-PAGE and Western blot revealed the presence of a mixture which consists of entire molecules ( trimers ), but also from polypeptides which are missing one or two of the monomer extracellular domains (dimers and monomers, respectively). This result indicates that the heterologous expression system which is used (P. pastoris), is suitable for the expression of functional bilateral concatamers (β2-24-α4-mECD), but is rather unsuitable for the expression of more complex structures, probably due to events of homologous recombination of the plasmids before or during integration into the yeast genome . Interestingly, deglycosylation of the concatamer did not affect its ligand binding properties. Furthermore, the deglycosylated β2-24-α4 ECD existed mainly in monomeric form, thus forming an appropriate material for structural studies and possibly for pharmacological evaluation of novel α4β2 nAChR-specific agonists.

No presente trabalho, cultivou-se o microrganismo Escherichia coli RR1, contendo o vetor que carrega o gene estrutural para a síntese do hormônio de crescimento humano (hGH) (baseado no promotor pL e pR do fago l sob controle do repressor termosensível cI857) em processos descontínuo e descontínuo-alimentado realizados em biorreatores com capacidade útil de 2 e 4 L. Tal cepa é auxotrófica com relação aos aminoácidos l-leucina e l-prolina e à tiamina (vitamina B1). Nos cultivos descontínuos com concentrações menores de extrato de levedura e bactotriptona em relação ao meio denominado basal, a concentração celular foi baixa, atingindo 2,4 g.L-1, com fator de conversão glicose à células de 0,25 g.g-1. Em cultivos descontínuos com aumento (em relação ao meio basal) da concentração de extrato de levedura e de bactotriptona e com adição de l-prolina, a concentração celular alcançou valores da ordem de 5,9 g.L-1 e fator de conversão glicose à células de 0,48 g.g-1, simultaneamente à maior formação de acetato (2,5 g.L-1), este último prejudicial ao processo. Contudo, este resultado de crescimento celular não se repetiu devido a mudança do lote de células utilizado entre o primeiro e o segundo conjunto de ensaios. Os cultivos descontínuos-alimentados foram realizados com diferentes formas de alimentação bem como diferentes composições de solução de alimentação. Uma alimentação contínua com velocidade exponencial e composição semelhante à do meio, pareceu ser a mais favorável, levando à concentração celular final de 9,2 g.L-1 e fator de conversão glicose a células, na fase descontínua-alimentada, de 0,36 g.g-1. Os ensaios com indução térmica não foram eficientes provavelmente devido à problemas na detecção das concentração de glicose existente no instante inicial da ativação da síntese do hGH. Esta glicose presente pode ter prejudicado a formação do hGH por conseqüência do processo fermentativo causado pelo aumento da temperatura e pela presença de elevada concentração de nutrientes complexos. O meio de cultivo utilizado possivelmente não supriu as necessidades metabólicas da célula para a síntese do hormônio de crescimento humano e em nenhum dos cultivos com indução térmica houve a produção de hGH. / In the present work, the host Escherichia coli RR1, having the vector with the structural gene for human growth hormone (hGH) synthesis, based on pL or pR promoters from bacteriophage l under the control of the thermosensitive repressor cI857, was cultivated in batch and fed-batch cultures in bioreactors with working volumes of 2 and 4 L. This host has amino acids (l-leucine and l-proline) and thiamine (Vitamin B1) auxotrophy. Batch cultures under low yeast extract and bacto-tryptone concentrations (relative to the basal medium) resulted in a low biomass yield (2.4 g.L-1) and cell yield on glucose (0.25 g.g-1). Increasing these concentrations and adding l-proline to the medium led to higher biomass formation (5.9 g.L-1), cell yield on glucose (0.48 g.g-1) and acetate high levels (2.5 g.L-1), which were harmful to the process. However, these results of cellular growth were not reproducible due to different cell stocks applied. The fed-batch cultures were performed under different feeding strategies and different nutrients concentrations of the feeding solution. A continuous exponential feeding rate with growth medium-like composition seemed to be the most favorable, reaching final cellular concentration of 9.2 g.L-1 and yield on glucose on fed-batch mode of 0.36 g.g-1. The heat-shock runs were not efficient probably due to problems in detection of glucose concentration existing on initial instant of hGH activation synthesis. Glucose interferes with the hGH synthesis because the fermentation caused by temperature shift and presence of high complex nutrients concentration. The culture medium used, probably was not able to supply cell metabolic needs for the human growth hormone synthesis and in no other temperature-induced experiment the hGH production was observed.

Neste trabalho, construiu-se linhagens recombinantes de Pichia pastoris e Saccharomyces cerevisiae que expressam hormônio de crescimento de carpa (GHc). Para expressão de GHc em P. pastoris, o cDNA de GH de H. molitrix foi inserido nos plasmídeos pHILD2 (expressão interna) e pPIC9K (expressão e secreção) dando origem aos plasmídeos pHILGH e pPICGH. A expressão em S. cerevisiae foi controlada pelo promotor e terminador PGK e o cassete de expressão ladeado por elementos δ, dirigindo a integração em múltiplas cópias no genoma. Ainda, construiu-se um plasmídeo onde a sequência de GHc foi fusionada a uma sequência sinal modificada (sequência sinal de proteína de K. lactis, fusionada a fragmento de interleucina humana), visando altos níveis de secreção. Os transformantes (expressão interna) expressaram proteína recombinante de 20 KDa. Um dos transformantes de S. cerevisiae secreta cerca de 1045,0 µg/ml de GHc. As proteínas recombinantes apresentaram hibridação específica contra anticorpo anti GHc comercial indicando que são biologicamente ativas. / In order to achieve the GHc expression in P. pastoris, the GH cDNA of the carp H. molitrix was introduced into the plasmids pHILD2 (intracellular expression) and pPIC9K (expression and secretion) originating the plasmids pHILGH and pPICGH. The expression of the GH cassette in S. cerevisiae was driven by the PGK promoter and terminator and the cassette was flanked by the δ elements, which provides multiple copies integration into the genome. We also constructed a plasmid in which the GHc cDNA sequence was fused to a modified signal sequence (K. lactis protein signal sequence fused to a human interleukin fragment), aiming to reach high levels of secretion. P. pastoris and S. cerevisiae recombinant clones (intracellular expression) were shown to express a recombinant protein of 20 KDa. One of the S. cerevisiae recombinant clones secreted around 1045.0 µg/ml of GHc. The recombinant proteins showed hybridization of the bands against antibody anti-commercial GHc. The results indicate that the recombinant proteins produced in this work are biologically active.

A descoberta das monoxigenases de polissacarídeos líticas dependentes de cobre (LPMOs), que agem em sinergismo com outras enzimas na desconstrução da celulose, gerou um grande interesse da comunidade científica por seu potencial de aplicação na produção de biocombustíveis a partir de resíduos lignocelulósicos. A busca por essas proteínas auxiliares em microrganismos surgiu como uma estratégia promissora, pois há grande diversidade de isoformas e disponibilidade de sequências genômicas dessas proteínas. Diante disso, o presente trabalho teve como objetivo expressar LPMOs recombinantes de origem bacteriana em levedura Komagataella phafii. Foram obtidos clones de K. phafii transformados com 6 genes selecionados a partir de banco de dados. Análises da cinética de expressão proteica por técnica de western blot indicaram secreção apenas da LPMO de 25 kDa codificada pelo gene de Thermobifida fusca YX. Ensaios de produção da LPMO de T. fusca foram realizados em biorreator de 1L e Erlenmeyers de 250 mL e 1000 mL. Nos ensaios em Erlenmeyers houve a detecção da proteína, confirmada por SDS-PAGE e western blot, acompanhado de um alto crescimento microbiano. No ensaio em biorreator não houve detecção da proteína de interesse e o crescimento microbiano foi baixo. Com a confirmação da expressão da LPMO nos ensaios em Erlenmeyers, estes foram parcialmente purificados e avaliados por gel de proteínas e western blot. Os resultados obtidos mostram que o sistema de expressão de K. phafii foi eficiente, expressando a LPMO de T. fusca. / The discovery of copper-dependent lytic polysaccharide monooxygenase (LPMOs), auxiliary proteins which act in synergy with other enzymes on the cellulose degradation, generated a great interest from the scientific community due to their potential application in biofuels production from lignocellulosic residues. The search for these auxiliary proteins in microorganisms has emerged as a promising strategy because there is a great diversity of isoforms and availability of genomic sequences. Thus, the present work aimed to express bacterial LPMOs in Komagataella phafii. Clones of K. phafii transformed with 6 genes selected from database were obtained. Analysis of protein expression kinetics by western blot technique showed accumulation only of the LPMO of 25 kDa coded by the gene from Thermobifida fusca YX. Production of LPMO from T. fusca was tested in 1 L bioreactor and 250 mL and 1000 mL Erlenmeyer flasks. In Erlenmeyer flasks experiments, there was protein detection, confirmed by SDS-PAGE and western blot, and a high microbial growth. In the bioreactor assay, there was no target protein detection, and microbial growth was low. After confirmation of LPMO expression in Erlenmeyer flasks assays, protein were partially purified and confirmed by protein gel and western blot. Results obtained showed that the expression system of K. phafii was effective, expressing the LPMO from T. fusca.

The primary objectives of this research were to demonstrate that: 1.) antibiotic resistant bacteria can promote the survival of antibiotic sensitive organisms when grown simultaneously as biofilms in antibiotics, 2.) community-level multiple antibiotic resistance of polymicrobial consortia can lead to biofilm formation despite the presence of multiple antibiotics, and 3.) biofilms may benefit plasmid retention and heterologous protein production in the absence of selective pressure. Quantitative analyses of confocal data showed that ampicillin resistant organisms supported populations of ampicillin sensitive organisms in steady state ampicillin concentrations 13 times greater than that which would inhibit sensitive cells inoculated alone. The rate of reaction of the resistance mechanism influenced the degree of protection. Spectinomycin resistant organisms did not support their sensitive counterparts, although flow cytometry indicated that GFP production by the sensitive strain was improved. When both organisms were grown in both antibiotics, larger numbers of substratum-attached pairs at 2 hours resulted in greater biofilm formation at 48 hours. For biofilms grown in both antibiotics, a benefit to spectinomycin resistant organism’s population size was detectable, but the only benefit to ampicillin resistant organisms was in terms of GFP production. Additionally, an initial attachment ratio of 5 spectinomycin resistant organisms to 1 ampicillin resistant organism resulted in optimal biofilm formation at 48 hours. Biofilms also enhanced the stability of high-copy number plasmids and heterologous protein production. In the absence of antibiotic selective pressure, plasmid DNA was not detected after 48 hours in chemostats, where the faster growth rate of plasmid-free cells contributed to the washout of plasmid retaining cells. The plasmid copy number per cell in biofilms grown without antibiotic selective pressure steadily increased over a six day period. Flow cytometric monitoring of bacteria grown in biofilms indicated that 95 percent of the population was producing GFP at 48 hours. This research supports the idea that ecological interactions between bacteria contribute to biofilm development in the presence of antibiotics, and demonstrates that community-level multiple antibiotic resistance is a factor in biofilm recalcitrance against antibiotics. Additionally, biofilms may provide an additional tool for stabilizing high copy number plasmids used for heterologous protein production.

No presente trabalho, cultivou-se o microrganismo Escherichia coli RR1, contendo o vetor que carrega o gene estrutural para a síntese do hormônio de crescimento humano (hGH) (baseado no promotor pL e pR do fago l sob controle do repressor termosensível cI857) em processos descontínuo e descontínuo-alimentado realizados em biorreatores com capacidade útil de 2 e 4 L. Tal cepa é auxotrófica com relação aos aminoácidos l-leucina e l-prolina e à tiamina (vitamina B1). Nos cultivos descontínuos com concentrações menores de extrato de levedura e bactotriptona em relação ao meio denominado basal, a concentração celular foi baixa, atingindo 2,4 g.L-1, com fator de conversão glicose à células de 0,25 g.g-1. Em cultivos descontínuos com aumento (em relação ao meio basal) da concentração de extrato de levedura e de bactotriptona e com adição de l-prolina, a concentração celular alcançou valores da ordem de 5,9 g.L-1 e fator de conversão glicose à células de 0,48 g.g-1, simultaneamente à maior formação de acetato (2,5 g.L-1), este último prejudicial ao processo. Contudo, este resultado de crescimento celular não se repetiu devido a mudança do lote de células utilizado entre o primeiro e o segundo conjunto de ensaios. Os cultivos descontínuos-alimentados foram realizados com diferentes formas de alimentação bem como diferentes composições de solução de alimentação. Uma alimentação contínua com velocidade exponencial e composição semelhante à do meio, pareceu ser a mais favorável, levando à concentração celular final de 9,2 g.L-1 e fator de conversão glicose a células, na fase descontínua-alimentada, de 0,36 g.g-1. Os ensaios com indução térmica não foram eficientes provavelmente devido à problemas na detecção das concentração de glicose existente no instante inicial da ativação da síntese do hGH. Esta glicose presente pode ter prejudicado a formação do hGH por conseqüência do processo fermentativo causado pelo aumento da temperatura e pela presença de elevada concentração de nutrientes complexos. O meio de cultivo utilizado possivelmente não supriu as necessidades metabólicas da célula para a síntese do hormônio de crescimento humano e em nenhum dos cultivos com indução térmica houve a produção de hGH. / In the present work, the host Escherichia coli RR1, having the vector with the structural gene for human growth hormone (hGH) synthesis, based on pL or pR promoters from bacteriophage l under the control of the thermosensitive repressor cI857, was cultivated in batch and fed-batch cultures in bioreactors with working volumes of 2 and 4 L. This host has amino acids (l-leucine and l-proline) and thiamine (Vitamin B1) auxotrophy. Batch cultures under low yeast extract and bacto-tryptone concentrations (relative to the basal medium) resulted in a low biomass yield (2.4 g.L-1) and cell yield on glucose (0.25 g.g-1). Increasing these concentrations and adding l-proline to the medium led to higher biomass formation (5.9 g.L-1), cell yield on glucose (0.48 g.g-1) and acetate high levels (2.5 g.L-1), which were harmful to the process. However, these results of cellular growth were not reproducible due to different cell stocks applied. The fed-batch cultures were performed under different feeding strategies and different nutrients concentrations of the feeding solution. A continuous exponential feeding rate with growth medium-like composition seemed to be the most favorable, reaching final cellular concentration of 9.2 g.L-1 and yield on glucose on fed-batch mode of 0.36 g.g-1. The heat-shock runs were not efficient probably due to problems in detection of glucose concentration existing on initial instant of hGH activation synthesis. Glucose interferes with the hGH synthesis because the fermentation caused by temperature shift and presence of high complex nutrients concentration. The culture medium used, probably was not able to supply cell metabolic needs for the human growth hormone synthesis and in no other temperature-induced experiment the hGH production was observed.

Neste trabalho, construiu-se linhagens recombinantes de Pichia pastoris e Saccharomyces cerevisiae que expressam hormônio de crescimento de carpa (GHc). Para expressão de GHc em P. pastoris, o cDNA de GH de H. molitrix foi inserido nos plasmídeos pHILD2 (expressão interna) e pPIC9K (expressão e secreção) dando origem aos plasmídeos pHILGH e pPICGH. A expressão em S. cerevisiae foi controlada pelo promotor e terminador PGK e o cassete de expressão ladeado por elementos δ, dirigindo a integração em múltiplas cópias no genoma. Ainda, construiu-se um plasmídeo onde a sequência de GHc foi fusionada a uma sequência sinal modificada (sequência sinal de proteína de K. lactis, fusionada a fragmento de interleucina humana), visando altos níveis de secreção. Os transformantes (expressão interna) expressaram proteína recombinante de 20 KDa. Um dos transformantes de S. cerevisiae secreta cerca de 1045,0 µg/ml de GHc. As proteínas recombinantes apresentaram hibridação específica contra anticorpo anti GHc comercial indicando que são biologicamente ativas. / In order to achieve the GHc expression in P. pastoris, the GH cDNA of the carp H. molitrix was introduced into the plasmids pHILD2 (intracellular expression) and pPIC9K (expression and secretion) originating the plasmids pHILGH and pPICGH. The expression of the GH cassette in S. cerevisiae was driven by the PGK promoter and terminator and the cassette was flanked by the δ elements, which provides multiple copies integration into the genome. We also constructed a plasmid in which the GHc cDNA sequence was fused to a modified signal sequence (K. lactis protein signal sequence fused to a human interleukin fragment), aiming to reach high levels of secretion. P. pastoris and S. cerevisiae recombinant clones (intracellular expression) were shown to express a recombinant protein of 20 KDa. One of the S. cerevisiae recombinant clones secreted around 1045.0 µg/ml of GHc. The recombinant proteins showed hybridization of the bands against antibody anti-commercial GHc. The results indicate that the recombinant proteins produced in this work are biologically active.