Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes: I. The cloning of the CVP 19 gene and construction of the intracellular expression vector pPIC3.5K-CYP19. II. The transformation of the yeast, Pichia pastoris with the constructed vector. III. The expression ofP450arom in Pichia pastoris. IV. The determination of enzyme activity and isolation of the protein from the Pichia pastoris cells. V. The expression of P450c 17 in Pichia pastoris. VI. The determination of kinetic constants for the conversion of progesterone to 170H-progesterone and 160H-progesterone by P450c17. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre uitdrukkingsplasmied, pPIC3.5K-CYPI9. II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied. III. Die uitdrukking van aromatase in Pichia pastoris. IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia pastoris. V. Die uitdrukking van P450c17 in Pichia pastoris. VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na 170H-progesteroon en 160H-progesteroon deur P450c17.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical cancer has been established by several epidemiology studies. Cervical cancer is the second most common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing countries where limited access to medical facilities hampers efforts to prevent and screen for HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major structural protein L1, which protect against the most common high-risk HPV-types, are currently available. The high cost and type specificity of these commercially available vaccines have necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and therefore a chimera was constructed that contains an epitope of L2 inserted within the L1 sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known to be excellent producers of recombinant proteins due to their strongly inducible promoters that allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula polymorpha have been employed in several studies for heterologous protein production and levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and can therefore be used to manufacture products for use in humans. In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on SAF yield in H. polymorpha which is in contrast to previous research. The highest production levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was that all the strains that produced SAF at detectable levels were equally efficient at the production of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in the P. pastoris strains as significantly higher cell densities were achieved during culturing of these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be used as hosts for the production of a broad-spectrum HPV vaccine. / AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel, belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF. SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat hulle vorm word algemeen aanvaar as veilig vir menslike gebruik. In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1 was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die produksie van ‘n wye spektrum HPV entstof. / The NRF and the Department of Microbiology for financial support

<p>The methylotrophic yeast, <i>Pichia pastoris </i>(recently reclassified as <i>spp. Komagatella</i>) has long been regarded as a useful host organism for the production of recombinant proteins, particularly when using the AOX system which utilizes methanol as both the inducing agent as well as the primary carbon source for growth and energy. Significant historical work has shown that growth rate and protein productivity can be correlated to methanol concentration. However, the relationship between oxygen and protein productivity are less consistent. While with many variations models having been developed and used for analyzing culture kinetics, these models have only been applied to methanol concentration. Furthermore, while results for methanol are fairly consistent, oxygen considerations have been far less consistent. </p> <p> </p> <p>This work presents various bi-variate models which includes considerations for growth and inhibition for both methanol and oxygen with this expanded model showing strong alignment to previous works to both oxygen and methanol data. While more work is necessary to fully confirm and validate which form of the bivariate model is most appropriate, this work provides a framework necessary to expand analysis to include oxygen considerations. This framework has the potential to be used to further inform selection of feeding methodology as well as direct investigations into metabolic studies. </p>

Os produtos de modificação de lipoproteínas de baixa densidade (LDL) como a subfração eletronegativa [LDL(-)] desempenham um importante papel na progressão da aterosclerose. O acúmulo massivo de LDL modificada captada por macrófagos resulta em células espumosas que liberam mediadores inflamatórios e contribuem para a aterogênese. O scFv (single chain fragment variable) é um fragmento de anticorpo recombinante que contém o sítio completo de ligação ao antígeno. Diante do papel da LDL(-) na aterogênese e da necessidade de novas intervenções terapêuticas que possam inibir o acúmulo de lipídeos em macrófagos, este trabalho objetivou a expressão do scFv anti-LDL(-) 2C7 em Pichia pastoris, bem como a avaliação do efeito deste fragmento de anticorpo sobre a formação de células espumosas em cultura de macrófagos RAW 264.7. O vetor inicial de expressão pPIgLE apresentava como estratégia de detecção e purificação o fusionamento com a proteína A. No entanto, a alta imunogenicidade da proteína A inviabilizaria o estudo da proteína de fusão em cultura de macrófagos, o que determinou a substituição da estratégia de purificação anterior pela cromatografia com resina de níquel através da inserção de hexahistidina na região C-terminal da proteína. A análise de sequenciamento confirmou a presença da inserção e das regiões determinantes de complementariedade. O cassete de expressão com hexahistidina foi inserido no vetor pPIgLE de P. pastoris e transformado na linhagem SMD1168 (Invitrogen®). Testes preliminares de expressão em pequena escala permitiram a análise entre sete clones diferentes, demonstrando uma banda correspondente ao peso molecular de 28 KDa em SDS-PAGE, confirmado por Western Blot. A separação do scFv 2C7 através de resina de níquel obteve uma proteína pura, conforme foi analisado em SDS-PAGE corado com prata. A afinidade do scFv 2C7 a 9 LDL(-) foi confirmada por Dot Blot. O ensaio de captação de LDL(-) demonstrou que o scFv 2C7 foi eficaz na redução de células espumosas e este efeito foi acompanhado pela diminuição na expressão gênica de CD36, TLR-4 e COX-2. Baseado nestes dados, o scFv 2C7 demonstra uma propriedade importante para uma futura intervenção terapêutica para a aterosclerose. / The modification products of low-density lipoprotein (LDL), as the electronegative subfraction [LDL(-)], play an important role in the progression of atherosclerosis. The massive accumulation of modified LDL uptake by macrophages results in foam cells that release inflammatory mediators and contribute to atherogenesis. The scFv (singlechain fragment variable) is a recombinant antibody fragment that contains the complete site antigen-binding. Considering the role of LDL(-) in atherogenesis and the need for new therapeutic interventions that may inhibit the accumulation of lipids in macrophages, this study aimed the expression of anti-LDL(-) 2C7 scFv in Pichia pastoris and the evaluation of the effect of this recombinant antibody fragment on foam cells formation in cultured RAW 264.7 macrophages. The pPIgLE expression initial vector presented as a strategy for detection and purification the fusion with protein A. However, the high immunogenicity of the protein impairs the study of the fusion protein in cultured macrophages, leading to the replacement of the previous strategy of purification by chromatography with nickel resin by inserting hexahistidine tag at the C-terminus of the protein. The sequence analysis confirmed the presence of insertion and the complementary determining regions. The expression cassete with hexahistidine was inserted into the pPIgLE vector of P. pastoris and transformed in the SMD1168 strain (Invitrogen®). Preliminary tests of expression in small-scale allowed the analysis of seven different clones, showing a band corresponding to the molecular weight of 28KDa on SDS-PAGE, confirmed by Western Blot. The separation of 2C7 scFv by the nickel resin yield a pure protein, as it was shown by SDS-PAGE stained with silver. The affinity of 2C7 scFv was confirmed by Dot Blot. The assay of LDL(-) uptake showed that the 2C7 scFv was effective in reducing foam cells and this effect was determined by the decrease in gene expression of CD36, TLR-4 and COX-2. Based on these data, the 2C7 scFv demonstrates an important property for future therapeutic intervention for atherosclerosis

Tse, Yuk Tin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-149). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The influenza virus --- p.1 / Chapter 1.1.1 --- Influenza NA and its inhibitors --- p.3 / Chapter 1.1.2 --- Follow-up on the use of NAIs --- p.9 / Chapter 1.2 --- Sources of NA for experimental studies --- p.11 / Chapter 1.2.1 --- Viral sources --- p.11 / Chapter 1.2.2 --- NA isolation --- p.12 / Chapter 1.2.3 --- Recombinant NA expressed in cell lines --- p.12 / Chapter 1.2.4 --- Glycosylation on NA functionality --- p.13 / Chapter 1.2.5 --- Recombinant NA expressed in yeast --- p.15 / Chapter 1.3 --- Research objectives --- p.16 / Chapter 2 --- Cloning of Influenza Neuraminidase and Expression in P. pastoris --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.1.1 --- Full-length cloning of the A/HongKong/483/97(H5N 1) NA --- p.17 / Chapter 2.1.2 --- Identification of the H274 equivalent --- p.19 / Chapter 2.1.3 --- The experiment --- p.22 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Preparation of chemically competent Escherichia coli --- p.23 / Chapter 2.2.1.1 --- Reagents --- p.23 / Chapter 2.2.1.2 --- Reagent setup --- p.23 / Chapter 2.2.1.3 --- Equipment --- p.23 / Chapter 2.2.1.4 --- Procedure --- p.23 / Chapter 2.2.2 --- Amplification of N1 NA and EGFP genes --- p.24 / Chapter 2.2.2.1 --- Reagents --- p.24 / Chapter 2.2.2.2 --- Reagent setup --- p.25 / Chapter 2.2.2.3 --- Equipment --- p.25 / Chapter 2.2.2.4 --- Procedure --- p.26 / Chapter 2.2.2.4.1 --- Amplification of the full-length N1 NA gene from cDNA --- p.26 / Chapter 2.2.2.4.2 --- Amplification of the EGFP gene --- p.26 / Chapter 2.2.3 --- TA cloning of PCR products --- p.27 / Chapter 2.2.3.1 --- Reagents --- p.27 / Chapter 2.2.3.2 --- Reagent setup --- p.27 / Chapter 2.2.3.3 --- Equipment --- p.28 / Chapter 2.2.3.4 --- Procedure --- p.28 / Chapter 2.2.3.4.1 --- TA cloning of PCR products --- p.28 / Chapter 2.2.3.4.2 --- Site-directed mutagenesis by overlapping PCR --- p.30 / Chapter 2.2.4 --- Construction of P. pastoris expression vectors --- p.31 / Chapter 2.2.4.1 --- Reagents --- p.31 / Chapter 2.2.4.2 --- Reagent setup --- p.31 / Chapter 2.2.4.3 --- Procedure --- p.32 / Chapter 2.2.4.3.1 --- Generation of N1 NA expression vectors --- p.32 / Chapter 2.2.4.3.2 --- Generation of EGFP expression vectors --- p.34 / Chapter 2.2.5 --- Transformation of P. pastoris --- p.37 / Chapter 2.2.5.1 --- Reagents --- p.37 / Chapter 2.2.5.2 --- Reagent setup --- p.37 / Chapter 2.2.5.3 --- Equipment --- p.38 / Chapter 2.2.5.4 --- Procedure --- p.38 / Chapter 2.2.5.4.1 --- Preparation and transformation of electrocompetent P. pastoris --- p.38 / Chapter 2.2.5.4.2 --- PCR analysis of P. pastoris transformants (colony PCR) --- p.39 / Chapter 2.2.6 --- Expression of N1 NA and EGFP in P. pastoris --- p.40 / Chapter 2.2.6.1 --- Reagents --- p.40 / Chapter 2.2.6.2 --- Reagent setup --- p.40 / Chapter 2.2.6.3 --- Procedure --- p.41 / Chapter 2.2.6.3.1 --- Small-scale protein expression in P. pastoris --- p.41 / Chapter 2.2.6.3.2 --- Sequence alignment --- p.42 / Chapter 2.2.6.3.3 --- Data processing --- p.42 / Chapter 2.3 --- Results and Discussion --- p.43 / Chapter 2.3.1 --- Cloning of NA and EGFP into the pPICZB expression vector --- p.43 / Chapter 2.3.2 --- Growth of P. pastoris transformants --- p.51 / Chapter 3 --- Physical Characterization of Influenza Neuraminidase Expressed in P. pastoris --- p.53 / Chapter 3.1 --- Background --- p.53 / Chapter 3.1.1 --- Structural significance of disulphide bonds in NA --- p.53 / Chapter 3.1.2 --- Localization of recombinant N1 NA in P. pastoris --- p.55 / Chapter 3.1.3 --- The experiment --- p.56 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Differential centrifugation --- p.57 / Chapter 3.2.1.1 --- Reagents --- p.57 / Chapter 3.2.1.2 --- Reagent setup --- p.57 / Chapter 3.2.1.3 --- Equipment --- p.57 / Chapter 3.2.1.4 --- Procedures --- p.58 / Chapter 3.2.1.4.1 --- Cell harvesting and lysis --- p.58 / Chapter 3.2.1.4.2 --- Preparation of crude membrane --- p.58 / Chapter 3.2.1.4.3 --- Preparation of plasma membrane --- p.58 / Chapter 3.2.2 --- Sodium dodecyl sulphate polyaciylamide gel electrophoresis (SDS-PAGE)… --- p.59 / Chapter 3.2.2.1 --- Reagents --- p.59 / Chapter 3.2.2.2 --- Reagent setup --- p.60 / Chapter 3.2.2.3 --- Equipment --- p.61 / Chapter 3.2.2.4 --- Procedure --- p.61 / Chapter 3.2.3 --- Immunoblotting --- p.61 / Chapter 3.2.3.1 --- Reagents --- p.61 / Chapter 3.2.3.2 --- Reagent setup --- p.62 / Chapter 3.2.3.3 --- Equipment --- p.62 / Chapter 3.2.3.4 --- Procedure --- p.62 / Chapter 3.2.3.4.1 --- Electroblotting --- p.62 / Chapter 3.2.3.4.2 --- Blocking and probing --- p.63 / Chapter 3.2.3.4.3 --- Immunodetection --- p.63 / Chapter 3.2.3.4.4 --- Molecular weight determination --- p.63 / Chapter 3.2.4 --- Confocal microscopy --- p.64 / Chapter 3.2.4.1 --- Equipment --- p.64 / Chapter 3.2.4.2 --- Procedure --- p.64 / Chapter 3.2.4.2.1 --- Image acquisition --- p.64 / Chapter 3.2.4.2.2 --- Image processing --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Localization of recombinant N1 NA in P. pastoris sub-cellular fractions --- p.66 / Chapter 3.3.2 --- Molecular weight determination for the N1 NA expressed in P. pastoris --- p.69 / Chapter 3.3.3 --- Cellular localization of recombinant N1 NA in P. pastoris --- p.71 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- Molecular weight determination for N1 NA expressed in P. pastoris --- p.77 / Chapter 3.4.2 --- Disulphide bond formation in N1 NA expressed in P. pastoris --- p.78 / Chapter 3.4.3 --- Cell-surface association of recombinant N1 NA in P. pastoris --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter 4 --- Functional Characterization of Influenza Neuraminidase Expressed in P. pastor --- p.is / Chapter 4.1 --- Background --- p.82 / Chapter 4.1.1 --- Fluorometric NA activity assay --- p.82 / Chapter 4.1.2 --- Colorimetric assay of NA activity --- p.84 / Chapter 4.1.3 --- The experiment --- p.85 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Fluorometric assay of N1 NA expressed in P. pastoris --- p.86 / Chapter 4.2.1.1 --- Reagents --- p.86 / Chapter 4.2.1.2 --- Reagent setup --- p.86 / Chapter 4.2.1.3 --- Equipment --- p.86 / Chapter 4.2.1.4 --- Procedure --- p.87 / Chapter 4.2.1.4.1 --- Calibrating cell density with viable cell counts --- p.87 / Chapter 4.2.1.4.2 --- End-point measurement of NA activity --- p.87 / Chapter 4.2.1.4.2.1 --- Determination of expression yield --- p.89 / Chapter 4.2.1.4.2.2 --- End-point assay of NAI sensitivity --- p.89 / Chapter 4.2.1.4.3 --- Kinetic measurement of NA activity --- p.90 / Chapter 4.2.1.4.3.1 --- Derivation ofV0 --- p.92 / Chapter 4.2.1.4.3.2 --- Graphical determination of KM --- p.93 / Chapter 4.2.1.4.3.3 --- Graphical determination of KI --- p.94 / Chapter 4.2.2 --- Colorimetric assay of N1 NA expressed in P. pastoris --- p.96 / Chapter 4.2.2.1 --- Reagents --- p.96 / Chapter 4.2.2.2 --- Reagent setup --- p.96 / Chapter 4.2.2.3 --- Equipment --- p.96 / Chapter 4.2.2.4 --- Procedure --- p.96 / Chapter 4.3 --- Results --- p.98 / Chapter 4.3.1 --- CFU determination --- p.98 / Chapter 4.3.2 --- Fluorescent NA activity assay for N1 NA expressed in P. pastoris --- p.98 / Chapter 4.3.2.1 --- End-point measurement of NA activity --- p.98 / Chapter 4.3.2.1.1 --- Course of N1 NA expression in P. pastoris --- p.102 / Chapter 4.3.2.1.1.1 --- NA activity per unit cell mass --- p.102 / Chapter 4.3.2.1.1.2 --- Yield of NA --- p.102 / Chapter 4.3.2.1.2 --- End-point assay for NAI sensitivity --- p.105 / Chapter 4.3.2.2 --- Kinetic measurement of NA activity and NAI sensitivity --- p.107 / Chapter 4.3.2.2.1 --- Graphical determination of KM --- p.107 / Chapter 4.3.2.2.2 --- Graphical determination of KI --- p.107 / Chapter 4.3.2.3 --- Colorimetric NA activity assay --- p.111 / Chapter 4.4 --- Discussion --- p.114 / Chapter 4.4.1 --- Fluorescent NA activity assay of N1 NA expressed in P. pastoris --- p.115 / Chapter 4.4.1.1 --- End-point measurement of NA activity --- p.115 / Chapter 4.4.1.1.1 --- Time course of expression --- p.115 / Chapter 4.4.1.1.2 --- Effect of H275Y mutation on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.1.3 --- Effect of C-terminal tags on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.2 --- Kinetic measurement of NA activity --- p.118 / Chapter 4.4.1.2.1 --- Graphical determination of KM --- p.119 / Chapter 4.4.1.2.2 --- Graphical determination of KI --- p.120 / Chapter 4.4.1.3 --- Comparison of fluorometric NA activity assays for use with whole P pastoris cells --- p.122 / Chapter 4.4.2 --- Colorimetric NA activity assay --- p.124 / Chapter 4.5 --- Conclusion --- p.126 / Chapter 5 --- Conclusions and Discussions --- p.127 / Chapter 5.1 --- General conclusions --- p.127 / Chapter 5.2 --- Follow-up --- p.127 / Chapter 5.2.1 --- Studies of influenza NA with enhanced activity --- p.128 / Chapter 5.2.2 --- NAI screening using yeast-expressed NA --- p.132 / Appendix --- p.134 / References --- p.141

O parvovírus B19 é o agente causador de eritemas infecciosos em crianças, hidropsia fetal em mulheres gestantes, esse vírus pode causar anemia crônica e crise aplástica transitória respectivamente. A levedura P. pastoris é um sistema de expressão usado na produção de várias proteínas heterólogas. O objetivo deste trabalho foi expressar as proteínas VP1 e VP2 do parvovírus humano B19 em levedura P. pastoris. As seqüências gênicas VP1 e VP2 foram amplificadas por PCR, usando DNA do vírus B19, os produtos obtidos foram inicialmente subclonados no vetor pGEM-TEasy. Os fragmentos de DNA foram digeridos com enzima de restrição EcoRI e NotI , purificados e inseridos no vetor de expressão e excreção pPIC9K de P. pastoris, entre os sítios EcoRI e NotI. Para expressão das proteínas recombinantes VP1 e VP2 de parvovírus humano B19, os transformantes foram crescidos em glicerol e induzidos pela adição de metanol. As expressões dos antígenos recombinantes foram analisadas por SDS-PAGE e atividade biológica foram confirmadas pelos ensaios imunológicos ELISA, Dot-Blot e Western Blot. / Human Parvovirus B19 is the causative agent of erythema infectiosum in children, hydrops fetalis in pregnant women, B19 may cause chronic anemia and aplastic crisis, respectively. The yeast P. pastoris expression system is being used for the production of various recombinant heterologous proteins. The objective of this work was to express the VP1 and VP2 proteins of the human parvovirus B19 in the yeast Pichia pastoris. The coding sequence of VP1 and VP2 were amplified by PCR, using DNA virus of B19. PCR-products were initially subcloned in the vector pGEM-TEasy. The DNA fragment EcoRI and NotI was excised, purified, and inserted between the sites EcoRI and NotI of P. pastoris expression-secretion vector pPIC9K.For heterologous expression of the proteins VP1 and VP2 Human parvovirus B19, the transformants were growth on glycerol and induced by the addition of methanol. The expressed recombinant antigens VP1 and VP2 were analyzed by SDS-PAGE and its biological activity were confirmed through Enzyme immunoassay EIA, Dot-Blot e Western Blot.

Plant secondary metabolites are essential for normal growth and development in plants ultimately affecting crop yield. They play roles ranging from appearance of the plants to defending against pathogen attack and herbivory. They have been used by humans for medicinal and recreational purposes amongst others. Glycosyltransferases catalyze the transfer of sugars from donor substrates to acceptors. Glucosyltransferases are a specific type of glycosyltransferases known to transfer glucose molecules from a glucose donor to a glucose acceptor (aglycone) producing the corresponding glucose secondary metabolite or glycone, in this case glucosides. It was hypothesized that SBIP68, a tobacco putative glucosyltransferase-like protein glucosylated salicylic acid. Salicylic acid is an essential plant defense secondary metabolite. SBIP68 was cloned and heterologously expressed in both prokaryotic and eukaryotic systems. Results from activity screening suggest that SBIP68 is a UDP-glucose flavonoid glucosyltransferase with broad substrate specificity. Further studies are required to fully characterize SBIP68.

Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase. The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced inEscherichia coli. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase. As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template. The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction. Keywords:apyrase, Pyrosequencing technology, Zbasictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,Pichia pastoris, Echerichia coli.

Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity. In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions. <b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity

Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl transfer reaction between a &gamma / -carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular microbial protransglutaminase (pro-MTG) producing recombinant Pichia pastoris strains by using genetic engineering techniques. In this context first,protransglutaminase gene (pro-mtg) from Streptomyces mobaraensis was amplified by PCR both for intracellular and extracellular constructs using proper primers then they were cloned into the pPICZ&alpha / -A expression vectors, separately. Both intracellular (pPICZ&alpha / A::pro-mtgintra) and extracellular (pPICZ&alpha / A::pro-mtgextra) constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Pichia pastoris X33 cells were transfected by linear pPICZ&alpha / A::pro-mtgintra and pPICZ&alpha / A::pro-mtgextra, separately and plasmids were integrated into the Pichia pastoris X33 genome at AOX1 locus. After constructing the recombinant P. pastoris strains, batch shaker bioreactor experiments were performed for each recombinant cell and the best producing strains were selected according to Dot blot and SDS-PAGE analyses. The selected recombinant P. pastoris strains, carrying pPICZ&alpha / A::promtgextra gene and pPICZ&alpha / A::pro-mtgintra gene in their genome were named as E8 and I1, respectively. Afterwards, a controlled pilot scale bioreactor experiment in a working volume of 1 L was performed with E8 clone and produced pro-MTG was activated by Dispase I. The variations in the recombinant MTG activity, cell concentration, total protease activity, AOX activity and organic acid concentrations throughout the bioprocess were analyzed and specific growth rates, specific consumption rates and yield coefficients were calculated regarding to measured data. Maximum MTG activity was obtained as 4448 U L- 1 and the maximum cell concentration was measured as 74.1 g L-1 at t=36 h of the bioprocess. In this study, an active transglutaminase enzyme was produced extracellularly by P. pastoris for the first time and the third highest extracellular MTG activity was achieved with E8 clone.