Orientador: Nilson Ivo Tonin Zanchin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T12:40:52Z (GMT). No. of bitstreams: 1 KosckyPaier_CarlosRoberto_M.pdf: 3760581 bytes, checksum: 99331529324819b59a4360d60efd9b9a (MD5) Previous issue date: 2009 / Resumo: A quinase de 70 kDa da proteína ribossomal S6, isoforma 1 (S6K1), é uma fosfoproteína implicada na regulação de genes relacionados ao controle da tradução em mamíferos e possui uma forma nuclear (a1) e uma citoplasmática (a2). A fosforilação do seu principal alvo, a proteína RPS6, tem sido comumente associada ao recrutamento seletivo dos 5'-TOP (5' tract of oligopyrimidine) mRNAs pela maquinaria de tradução, embora haja estudos contrariando esta hipótese. Devido às funções de seus demais alvos, S6K1 tem sido implicada na sobrevivência celular e em diversos outros processos, como crescimento, câncer e resistência à insulina. S6K1 é ativada por um mecanismo que envolve fosforilação seqüencial através da ativação das vias mTORC1 (complexo 1 do alvo da rapamicina em mamíferos) e PI3K (fosfoinositol-3 quinase). Como uma quinase da família AGC, S6K1 deve ser fosforilada por mTORC1 no resíduo Thr389 do domínio hidrofóbico e, em seguida, por PDPK1 (proteína quinase 1 dependente de fosfoinositol) no resíduo Thr229 da alça T do domínio catalítico. Estes eventos ocorrem somente após a fosforilação em diversos sítios do domínio auto-inibitório carboxiterminal, por mTORC1. O objetivo deste trabalho foi desenvolver um ensaio modelo para análise da função da S6K1 in vitro e utilizá-lo como ferramenta na elucidação do papel de proteínas adaptadoras da via de mTOR em interações com a S6K1. Para isso foi necessário produzir as proteínas recombinantes para ensaios de interação e para realização de um ensaio de atividade para a S6K1. Foram testados vários sistemas de expressão para Escherichia coli para produção das construções GST-S6K1a1-His6, GST-S6K1a2-His6 e GST-S6K1a2T389E?CT (forma a2 de S6K1 com a substituição T389E e o carboxiterminal truncado), GST-PDPK1 e GST-CDPDPK1 (domínio catalítico de PDPK1 fusionado a GST). A expressão das formas truncadas de S6K1 e PDPK1 foi mais eficiente em E. coli. Embora o rendimento tenha ficado muito aquém do esperado, foi suficiente para os ensaios de interação in vitro. Também foi feita a expressão em E. coli da região C-terminal da proteína RPS6, que é o substrato da S6K1, em fusão com a proteína D do fago ?. Posteriormente, foram montados sistemas de expressão das construções His6-S6K1a2T389E?CT e His6-CDPDPK1 em células de inseto, a partir de vetor de baculovírus. Constatou-se que essas construções são expressas na forma de fosfoproteínas em células de inseto. Ensaios de GST pull-down com GST-S6K1a2-His6 e GST-S6K1a2T389E?CT contra as duas isoformas da subunidade catalítica da PP2AC, His6-PP2ACa(maior) e His6-PP2ACa(menor), revelaram que His6-PP2ACa(maior) não interage com GST-S6K1a2-His6, embora interaja fortemente com GST-S6K1a2T389E?CT. Já a construção His6-PP2ACa(menor) interage fracamente com as construções GST-S6K1a2-His6 e GST-S6K1a2T389E?CT. Tomados em conjunto, os resultados sugerem que a presença do C-terminal não fosforilado de S6K1a2 impede a interação com PP2ACa(maior). PP2ACa(menor) comporta-se de forma completamente diferente da isoforma maior, pois a interação entre PP2ACa(menor) e S6K1a2 parece ser independente do carboxiterminal da quinase, visto que as quantidades de S6K1a2T389E?CT e de S6K1a2 inteira que interagem com PP2ACa(menor) são semelhantes. Esses resultados necessitam ainda serem confirmados in vivo. Outros experimentos de GST pull-down confirmaram que as construções de S6K1 não interagem com a4, embora interajam com TIPRL1. Se confirmado in vivo, esse resultado compõe um novo quadro na regulação coordenada entre mTOR1 e PP2A, do qual TIPRL1 parece participar. As construções genéticas e os sistemas de expressão gerados neste trabalho possibilitaram a obtenção dos reagentes necessários para analisar o mecanismo de regulação da quinase S6K1, mediado por proteínas regulatórias. Permitem também desenvolver uma série de experimentos, como busca de inibidores específicos para a S6K1, que dependem da reconstituição de ensaios de atividade in vitro com a S6K1 ativada. Contudo, o ensaio de atividade realizado não apresentou resultados satisfatórios e precisa ser desenvolvido. / Abstract: The 70kDa ribosomal S6 protein kinase 1 (S6K1) is a phosphoprotein involved in the regulation of genes related to translational control in mammals. S6K1 shows distinct nuclear (a1) and cytoplasmic (a2) forms. Phosphorylation of the S6K1 best characterized target, the protein of the small ribosomal subunit (RPS6), has been generally associated to the selective recruitment of the 5'-TOP mRNAs (5' tract of oligopyrimidine) by the translational machinery, although there is still some controversy on this issue. Due to the function of its targets, S6K1 has been implicated in several cellular processes including cell growth, cancer and insulin resistance. S6K1 is activated by a mechanism of sequential phosphorylation following activation of the mTORC1 (mammalian target of rapamycin complex 1) and PI3K (phosphoinositide-3-kinase) pathways. As a kinase of the AGC family, S6K1 activation requires mTORC1 phosphorylation of residue Thr389 of the hydrophobic domain followed by PDPK1 (phosphoinositide dependent protein kinase 1) phosphorylation of residue Thr229 at the T loop of the catalytic domain. These take place only after phosphorylation by mTORC1 of several residues of the autoinhibitory C-terminal domain. The objective of this work was to develop an assay to analyze the function of S6K1 in vitro and use it as a tool in the discovering of the functions of regulators proteins of the mTOR cascade in interactions with S6K1. For these purposes, expression systems were constructed to produce the various recombinant proteins to be used in the interaction and activity assays. Several genetic constructions were tested in Escherichia coli for the production of GST-S6K1a1-His6, GST-S6K1a2-His6 and GST-S6K1a2T389E?CT (a2 form of S6K1 with the T389E substitution and truncated carboxiterminus), GST-PDPK1 and GST-CDPDPK1 (GST fusion protein of the catalytic domain of PDPK1). The truncated forms were expressed more efficiently in E. coli. Although the yield in E. coli was lower than expected, it was sufficient to perform interaction assays. The C-terminal domain of RPS6, a substrate for S6K1, was successfully expressed in E. coli as a fusion protein with the phage ? protein D. Subsequently, expression systems for production of His6-S6K1a2T389E?CT and His6-CDPDPK1 in insect cells were constructed using baculovirus vectors. It was found that these constructs are expressed in the form of phosphoproteins in insect cells. GST pull-down assays using GST-S6K1a2-His6 e GST-S6K1a2T389E?CT to test interaction with the PP2AC isoforms His6-PP2ACa(major) and His6-PP2ACa(minor) revealed that His6-PP2ACa(major) does not interact with GST-S6K1a2-His6, although it interacts strongly with GST-S6K1a2T389E?CT. On the other hand, His6-PP2ACa(minor) interacts weakly with both GST- S6K1a2-His6 and GST-S6K1a2T389E?CT. This finding suggests that the unphosphorylated C-terminal of S6K1a2 inhibits interaction with PP2ACa(major). His6-PP2ACa(minor) behaves differently form His6-PP2ACa(major). Its interaction with S6K1a2 seems to be independent of the C-terminal since the amounts of S6K1a2T389E?CT and S6K1a2 that interact with His6-PP2ACa(minor) are similar. Future work in vivo is required to confirm these results. GST pull-down assays confirmed that a4 does not interact with the constructions of S6K1, while TIPRL1 interacts with them. If confirmed in vivo, these results provides a new perspective for the coordinated regulation between mTOR1 and PP2A, which apparently involves also TIPRL1. The genetic constructions and expression systems established in this work allow the production of the reagents required to study the mechanism of S6K1 regulation mediated by adaptor proteins. They will also allow the development of experiments such as screening for specific S6K1 inhibitors, which depend on reconstitution of S6K1 activity assays using activated S6K1. Nevertheless, the activity assay performed did not yield satisfactory outcomes and must be improved. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Neste trabalho foram estudados diversos métodos cromatográficos para a purificação de fatores recombinantes de coagulação VII (FVIIr) e VIII (FVIIIr) derivados de linhagens celulares humanas SK-Hep. O FVIIIr é utilizado para o tratamento da Hemofilia A, enquanto o FVIIr é utilizado para o tratamento da Hemofilia B e também a Hemofilia A. Produzir estes fatores em linhagens celulares humanas faz com os padrões de glicosilação, sulfatação e enovelamento destas proteínas sejam extremamente parecidos com os fatores endógenos produzidos no organismo humano. A purificação do FVIIIr através de técnicas de cromatografia multimodais usando a resina CaptoMMC, afinidade usando a resina FVIIISelect e troca iônica (SP-Sepharose) permitiu obter um produto bastante homogêneo e com perfil de banda (por SDS-PAGE) bem definido que demonstrou a presença esperada das cadeias leve e pesada (o Westen-Blott indicou que os anticorpos comerciais reconheceram a cadeia pesada da molécula estudada). As técnicas permitiram uma alta reprodutibilidade do processo onde sequencias de purificação indicaram o mesmo comportamento de perfis cromatográficos e o processo eliminou 99.5% ± 0,5% de proteínas inespecíficas, recuperando até 64% de FVIIIr. O FVIIr foi purificado com apenas uma única técnica cromatográfica usando a resina FVIISelect que isolou a proteína de interesse eliminando cerca de 99% de impurezas, recuperando praticamente todo o produto. O eluido da cromatografia de afinidade foi dialisado em membranas de 5 kDa o que resultou no processo de auto ativação da molécula de FVIIr, resultando em um aumento de sinal de até 5x em relação a quantidade inicial. O gel de SDS-PAGE e o Westen-Blott comprovaram o processo de auto-ativação no qual uma migração de banda de 50 kDa para 30kDa foi observada e os anticorpos comerciais contra FVII foram capazes de detecta-la. O método de purificação também foi bastante reproduzível e o perfil de banda muito semelhante se comparado ao produto comercial existente no mercado. Sendo assim, foi possível obter plataformas de purificação para as proteínas FVIIr e FVIIIr. / In this work, several chromatographic methods were studied for the purification of recombinant clotting factors VII (FVIIr) and VIII (FVIIIr) derived from human SK-Hep cell lines. The FVIIIr is used for the treatment of Hemophilia A, while the FVIIr is used for the treatment of Hemophilia B and Hemophilia A. Producing these factors in human cell lines results in glycosylation, sulphation and folding patterns similar to the endogenous factors produced in the human organism. Purification of FVIIIr by multimodal chromatography techniques using CaptoMMC resin, affinity using FVIIISelect resin and ion exchange (SP-Sepharose) yielded a fairly homogeneous and well-defined band profile (by SDS-PAGE) which demonstrated the expected presence of the light and heavy chains, Westen-Blott indicated that commercial antibodies recognized the heavy chain of the studied molecule. The techniques allowed a high reproducibility of the process where purification sequences indicated the same behavior of chromatographic profiles and the process eliminated 99.5% ± 0.5% nonspecific proteins and recovering up to 64% FVIIIr. FVIIr was purified with only a single chromatographic technique using the FVIISelect resin which isolated the protein by removing about 99% impurities and recovering virtually the entire product. The affinity chromatography eluate was dialyzed on 5 kDa membranes which resulted in the autoactivation process of the FVIIr molecule resulting in a signal increase of up to 5 fold over the initial amount. The SDS-PAGE gel and Westen-Blott demonstrated the auto-activation process where a migration of 50 kDa to 30 kDa band was observed and the commercial antibodies against FVII were able to detect the band. The purification method was also quite reproducible and the band profile very similar compared to the commercial products. Thus, it was possible to obtain purification platforms for the FVIIr and FVIIIr proteins.

Le polymorphisme génétique du virus de l’hépatite B (VHB) a déjà été étudié pourtenter de comprendre les facteurs viraux influençant l'évolution de la maladie, mais les étudessont discordantes. Ceci peut être lié au fait que les précédents travaux n’ont été menés quedans des populations avec une faible variété de génotypes et présentant des charges viralesplasmatiques (CVP) élevées.Nous avons donc étudié la variabilité du génome complet du VHB chez 422 individusinfectés chroniquement, naïfs de traitements anti-viraux et dont 38% présentaient une CVPinférieure à 103 UI/mL. L’optimisation de l’amplification par PCR du génome complet duVHB nous a permis de séquencer en technique Sanger plus de 90% du génome pour 320échantillons. Le séquençage direct a mis en évidence des co-infections. Ceci a été confirmé enséquençage clonal par pyroséquençage de 27 échantillons qui a montré des proportions departicules défectives variables mais toujours en co-infections avec des sous-populationssauvages. Le génotypage des séquences obtenues par technique Sanger a montré une grandereprésentativité des génotypes les plus fréquents (A à E) ainsi que 60 potentiels recombinantsinter-génotypiques. Cependant le séquençage clonal par pyroséquençage et clonage vectorielclassique de ces derniers montre la présence de co-infections de plusieurs génotypes ou laprésence de génotypes intermédiaires entre génotypes proches. Ceci est en défaveur derecombinaison par échange de matériel génétique comme ce qui a été suggéré dans lalittérature.Cette étude sera complétée par l’analyse de corrélation entre les polymorphismes et lesmarqueurs de mauvaise évolution de la pathologie. / The genetic polymorphism of hepatitis B virus (HBV) has been investigated tounderstand its impact on disease evolution, with discordant results. This could be due to thenarrow range of genotype and plasmatic viral load in these studies.We analysed complete genome variability of circulating HBV, in 422 chronicallyinfected patients. All were naive of anti-viral treatement and 38% had a plasmatic viral loadbelow 103 UI/mL. After optimisation of full length genome PCR amplification, we obtainedSanger sequences for more than 90% of HBV genome in 320 samples. We detected by directsequencing multiples co-infections that were confirmed by clonal pyrosequencing in 27samples. Defective viruses were always observed in co-infection with wild type virus. Directsequences showed a large representation of the most frequent genotypes (A to E), but also 60potential inter-genotypic recombinants. Clonal pyrosequencing and vectorial sequencingshowed that these potential recombinants were co-infections with different genotypes orintermediate genotypes located between close genotypes. These observations are incontradiction with the hypothesis described in the literature on recombination by geneticmaterial exchange.This study will be completed by a correlation analysis between the polymorphisms andmarkers of bad prognosis during HBV-induced disease.

Master of Science / Department of Grain Science and Industry / Praveen V. Vadlani / Synthesis of chemical derivatives from finite fossil fuels requires considerable energy inputs and leaves an undesirable environmental footprint. The emerging biorefinery approach leads to sustainable processing of biomass into a wide spectrum of bio-based products, catering to food, feed, chemicals, materials, and bioenergy industries. Cyanophycin (multi-L-arginyl-poly-L-aspartic acid, CGP) is a non-ribosomally synthesized reserve polypeptide, which consists of equimolar amounts of arginine and aspartic acid arranged as a polyaspartate backbone and arginine as the side chain. Cyanophycin is a source of the constituent N-functionalized platform chemical, which can be further processed into many other chemicals of importance. It can be hydrolyzed in mild condition to its constituent amino acids - aspartic acid and arginine. These amino acids may be utilized directly in food and pharmaceutical applications. Based on the chemical structure of these amino acids and the presence of functionalized nitrogen-containing groups, it is conceivable that a number of industrial chemicals can be synthesized, for example: 1, 4-butanediamine， a co-monomer in the production of nylon-4, 6. Other chemicals which could be obtained from cyanophcyin, that are currently prepared from fossil resources, include 1,4-butanediol and urea. Cyanophycin can also be hydrolyzed to a derivative with reduced arginine content or even to poly-aspartic acid, and used as a biodegradable substitute for synthetic polyacrylate in various technical process, such as water treatment (water softeners) and plastics. Cyanophycin is produced by most cyanobacteria in nature; however, these microbes are not suitable for large-scale production due to slow growth and low polymer content. Biosynthesis of cyanophycin is catalyzed by a single enzyme - cyanophycin synthetase (CphA), which is encoded by cyanophycin synthetase structure gene (cphA). The cphA gene can be expressed in several bacteria and plants. E.coli is one of the most commonly used bacterial hosts for the production of recombinant proteins. The recombinant culture has the ability to produce considerably large quantities of cyanophycin in a shorter period of time compared to cyanobacteria. Genome of Anabaena variabilis ATCC 29413 has been sequenced, and it contains the structural gene (cphA) for cyanophycin synthetase. The native enzyme-cyanophycin synthetase from this cyanbacterium culture had been purified and it consists of identical subunits of 98kDa. Polymerization of the amino acids to cyanophycin in vitro is dependent on the presence of ATP, K+, Mg2+, a (cyanophycin) primer and a thiol reagent such as β-mercaptoethanol in the reaction mixture. Our research is the first time that cphA gene from A. variabilis ATCC 29413 was cloned into E.coli. The 2.7 kb cphA gene was amplified by PCR, ligated to the vector pET45b+ and cloned into BL21 (DE3) pLysS and BL21 (DE3). Characterization of cyanophycin was performed by SDS-PAGE, HPLC, mass spectrometry and amino acid analysis. Results showed that protein with molecular weight of 21.5 to 31 kDa did not match any E.coli proteins when compared with E.coli protein data base, thereby showing expression of a protein foreign to host strain. Amino acids analyses showed that the cyanophycin produced by recombinant E.coli contained aspartic acid and arginine, and small amount of lysine, in the ratio: 1.05: 1: 0.2 (mass basis), thereby confirming cyanophycin biosynthesis. Experiments for high cyanophycin synthesis was performed at shake flask and 2-L fermentor level using recombinant BL21 (DE3) pLysS, LB broth as carbon and nutrient source, and casamino acids as primer. The maximum yield of cyanophycin obtained in flask level was 7.6% of cell dry mass, and the yield increased to 12.6% of cell dry mass at 2-L fermentor level. Cyanophycin is also referred to as “structural granules” because of substructures visible in electron micrographs. Phase contrast photomicrograph was able to depict cyanophycin inclusions in the cytoplasm, and transmission electron microscopy depicted finer details inside cell after IPTG induction.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: QUANTITATIVE YEAST PHYSIOLOGY AND NITROGEN METABOLISM DURING HETEROLOGOUS PROTEIN PRODUCTION By Johann F. Görgens The physiology and nitrogen metabolism of the yeast, Saccharomyces cerevisiae, during heterologous xylanase production in a defined medium was quantified by the comparison of isogenic yeast strains, whereby several potential limitations in the production of the heterologous xylanase could be identified. The presence of global sensing and regulatory mechanisms, by which the yeast is able to actively regulate both heterologous gene expression and the physiological response to the process, was also investigated. The deleterious effects of heterologous xylanase production on the physiology of the recombinant host were disproportionately large with respect to the amount of foreign protein produced. The cellular processes involved in this response were identified by the transcriptional profiling of isogenic recombinant strains, in a novel analytical approach to investigating foreign protein production by S. cerevisiae. Heterologous gene expression affected a combination of cellular processes and induced the yeast stringent stress response. The corresponding loss of metabolic functionality resulted in the disproportionate physiological effects of foreign protein production, similar to previous observations in recombinant Escherichia coli, and a possible reduction in attainable production levels. Reducing the propensity of recombinant gene expression to introduce metabolic stress may therefore increase production levels of foreign proteins by yeast. The metabolic vitality of transformed strains was also reduced by the presence of multiple copies of active, plasmid-based PGK1-promoters in the cell without expression of the heterologous gene. The negative effect was caused by an increase in the biosynthetic and glycolytic capacity of the strain at the expense of other processes. Production levels of heterologous xylanase were influenced by expression vector selection and the presence of auxotrophic mutations in transformed strains of S. cerevisiae. The increased transcription levels obtained with the multicopy plasmidbased YEp-type expression system, compared to the integrative YIp-type expression system, resulted in higher levels of xylanase production. Heterologous xylanase production thus did not saturate the secretory capacity of the host strain. The genetic stability of the autoselective YEp-type expression system in long-term chemostat culture was also demonstrated. High levels of heterologous xylanase production by transformed S. cerevisiae strains containing auxotrophic markers required the stabilisation of nitrogen metabolism via saturation of yeast cells with an excess of imported amino acids. By the removal of excessive auxotrophic markers, high levels of xylanase production by a prototrophic transformant in defined medium without amino acid addition could be obtained. Heterologous xylanase production by the prototrophic transformant was further enhanced by increasing the availability of preferred amino acids or succinate in the defined medium, indicating an additional requirement for metabolic precursors and building blocks for foreign protein synthesis. Comparable levels of heterologous xylanase production were obtained in high cell density cultures of the alternative yeast, Pichia stipitis, by the proper induction of the native ADH2-promoter, the control of oxygenation, and addition of an amino acid mixture to the defined medium, indicating the presence of generic limitations in transcription, nutrient availability and the yeast biosynthetic capacity for foreign protein production by various yeasts. The presence of global sensing and regulatory mechanisms was confirmed by the physiological response of S. cerevisiae to heterologous protein production, which included the downregulation of biosynthesis and growth, and the induction of various processes involved in the stringent stress response. Additionally, heterologous xylanase production was actively regulated on a posttranscriptional level by the auxotrophic transformants in response to the level of amino acid availability. The biosynthetic capacity for foreign protein production by both recombinant S. cerevisiae and P. stiptis was also regulated in response to the physiological state of the yeast and the availability of nutrients. The presence of these regulatory mechanisms complicated the manipulation of cellular biosynthesis at will. / AFRIKAANSE OPSOMMING: KWANTITATIEWE GIS-FISIOLOGIE EN -STIKSTOF METABOLISME GEDURENDE HETEROLOË PROTEÏEN PRODUKSIE Deur Johann Ferdinand Görgens Die fisiologie en stikstof-metabolisme van die gis, Saccharomyces cerevisiae, gedurende heteroloë xilanase produksie in ‘n gedefiniëerde medium is gekarakteriseer deur isogeniese gis-rasse te vergelyk, waardeur verskeie moontlike beperkings in die produksie van die heteroloë xilanase uitgewys kon word. Die teenwoordigheid van globale sensoriese- en beheer-meganismes, wat die gis in staat stel om beide heteroloë geen uitdrukking en die fisiologiese respons op die proses aktief te reguleer, is ook ondersoek. Die nadelige effekte van heteroloë xilanase produksie op die fisiologie van die rekombinante gasheer-organisme was uitermatig groot in vergelyking met die hoeveelheid vreemde proteïen wat geproduseer is. Die sellulêre prosesse verantwoordelik vir hierdie respons is identifiseer deur die transkripsionele profiele van isogeniese rekombinante rasse te vergelyk, in ‘n nuwe analitiese benadering tot die bestudering van vreemde proteïen produksie deur S. cerevisiae. Heteroloë geen uitdrukking het ‘n kombinasie van sellulêre prosesse geaffekteer en die gis se algemene voedingstres-respons geaktiveer. Die gepaardgaande verlies aan metaboliese funksie het die uitermatige fisiologiese effek van vreemde proteïen produksie veroorsaak, soortgelyk aan vorige waarnemings met rekombinante Escherichia coli. Die haalbare produksie-vlakke is moontlik ook verlaag deur hierdie respons. ‘n Verlaging van die geneigdheid van rekombinante geen uitdrukking om metaboliese stres te veroorsaak, mag dus die produksievlakke van vreemde proteïene in gis verbeter. Die metaboliese groei-potensiaal van die getransformeerde rasse is ook verlaag deur die teenwoordigheid van etlike aktiewe kopieë van plasmied-gebaseerde PGK1-promotors in die sel, sonder uitdrukking van die heteroloë geen, deur ‘n toename in die biosintetiese en glikolitiese kapasiteit ten koste van die ander sellulêre prosesse. Die produksievlakke van heteroloë xilanase is deur die keuse van uitdrukkings-sisteem en die teenwoordigheid van autotrofiese mutasies in die getransformeerde rasse van S.cerevisiae beïnvloed. Die verhoogde transkripsie vlakke wat met die multi-kopie, plasmied-gebaseerde YEp-tipe uitdrukkingsisteem, eerder as die geïntegreerde YIp-tipe sisteem, verkry is, het tot verhoogde xilanase produksie gelei. Heteroloë xilanase produksie het dus nie die uitskeidingskapasiteit van die gasheer versadig nie. Die genetiese stabiliteit van die autoselektiewe, YEp-tipe uitdrukkingsisteem in langtermyn chemostaat-kulture is ook gedemonstreer. Hoë vlakke van xilanase produksie deur getransformeerde S. cerevisiae rasse met autotrofiese merkers het die stabilisering van die stikstof metabolisme, deur die versadiging van die sel met ingevoerde aminosure, vereis. Die verwydering van oormatige autotrofiese merkers het tot hoë vlakke van xilanase produksie deur die prototrofiese transformant in gedefinieerde medium sonder aminosuur byvoeging gelei. Heteroloë xilanase produksie deur die prototrofiese transformant kon verder verbeter word deur die byvoeging van voorkeur-aminosure of suksinaat tot die gedefinieerde medium, en ‘n addisionele behoefte aan metaboliese voorloper-molekules en bou-blokke vir vreemde proteïensintese het dus bestaan. Vergelykbare vlakke van heteroloë xilanase produksie is in kulture met hoë sel-digthede van die alternatiewe gis, Pichia stipitis, verkry deur die doeltreffende induksie van die eiesoortige ADH2-promotor en die byvoeging van ‘n aminosuur-mengsel tot die gedefinieerde medium, wat die teenwoordigheid van generiese beperkinge in transkripsie, voedingstof-beskikbaarheid en biosintetiese kapasiteit van die gis vir vreemde proteïen produksie deur verskeie giste uitgewys het. Die teenwoordigheid van globale sensoriese- en beheer-meganismes is bevestig deur die fisiologiese respons van S. cerevisiae tot heteroloë proteïen produksie, wat die afwaartse regulering van biosintese en groei, en die induksie van verskeie prosesse betrokke by die algemene voedingstres-respons, ingesluit het. Heteroloë xilanase produksie is ook op ‘n na-transkripsionele vlak aktief gereguleer deur die autotrofiese transformante in reaksie tot die vlak van aminosuur beskikbaarheid. Die biosintetiese kapasiteit vir vreemde proteïen-produksie van beide rekombinante S. cerevisiae en P. stipitis is ook in reaksie tot die fisiologiese toestand van die gis en die beskikbaarheid van voedingstowwe gereguleer. Die teenwoordigheid van hierdie regulatoriese meganismes het die willekeurige manipulasie van sellulêre proteïen-biosintese bemoeilik.

Expressing foreign proteins in E.coli is a major challenge because they often tend to develop into unsolvable and inactive proteins. They aggregate into so called  inclusion bodies which prevent expression of the protein. This problem might be avoided by fusing the gene of the foreign protein with a soluble protein called solubility tags, which  function is to enhance the solubility of the foreign protein. This report investigates whether Sterol Carrier Protein-2 (SCP-2) could function as a solubility tag. The experiment was carried out by fusing SCP-2 to two recombinant proteins, Green fluorescent protein (GFP) and a form of chloroamphenicol acetyl transferase (CATΔ9). The gene fusion was then inserted into a pET-15 vector and transformed into  the E.coli strain BL21(DE3) to be expressed. The results obtained from Western blot and PageBlue staining indicates that SCP-2 does not enhance the solubility of GFP or CATΔ9 since neither of them was expressed.  Furthermore, previous studies have shown that GFP can in fact be expressed  usingmaltose binding protein (MBP) as a solubility tag. Unfortunately, no success has been made regarding CATΔ9. In conclusion, regarding the results from this report, SCP-2 does not function as a solubility tag. However, further studies should be carried out on SCP-2 with more experiments before rejecting the possibility to use SCP-2 as a solubility tag.

Ferric ABC Transporters. Pathogenic bacteria have evolved specialised iron acquisition systems that allow them to effectively colonise a host. One of these systems is the ferric binding protein (Fbp) complex that is a member of the ATP-Binding Cassette (ABC) superfamily of small molecule transporters. The Fbp complex is made up of three-components (FbpABC) that transports ferric iron from the periplasm to the cytoplasm of many Gram negative bacteria. FbpA binds iron in the periplasm and transports it to the FbpB transporter complex that permeates the cytoplasmic membrane. Here the iron is actively transported by FbpB through the membrane that is powered by ATP hydrolysis catalysed by FbpC, the cytoplasmic ATPase. Burkholderia cenocepacia is an opportunist pathogen that colonises the lungs of cystic fibrosis patients and is particularly resistant to antibiotic treatment. In this study the iron uptake system of B. cenocepacia strain J2315 is investigated. A putative FbpA from B. cenocepacia J2315 was expressed in the periplasm of Escherichia coli cells and the recombinant FbpA B. cenocepacia protein purified. The structural and electrochemical properties of native FbpA B. cenocepacia were investigated using UV Visible spectroscopy, spectro-electrochemistry, mass spectrometry and crystallographic techniques. It appears that FbpA B. cenocepacia is a novel member of the FbpA superfamily that selectively utilises citrate as an exogenous anion in ferric iron co-ordination. This is the first instance that a recombinant ferric binding protein has been documented as preferentially utilising citrate in this manner. The putative ATPase from B. cenocepacia (FbpC B. cenocepacia) was also expressed in E. coli but it was found to be insoluble. A number of expression systems were tested but none were found to be successful in generating sufficient quantities of FbpC B. cenocepacia for structural studies. Human β-defensin 2. Despite daily contact with a range of microorganisms, mammals do not regularly succumb to pathogenic invasion. One reason is the presence of an important defence mechanism uses a reservoir of antimicrobial peptides (AMPs) that are expressed in eukaryotes as a means of innate immunity. The AMP superfamily is composed of over 900 members, displays broad structural and sequence diversity and is active against a wide range of bacteria, fungi and viruses. β-defensins are small (3-5 kDa), cationic peptides that display antimicrobial activity against a range of microbes and have also been shown to act as chemo-attractants (chemokines) within the adaptive immune system. In this study we obtained milligram amounts of pure human β-defensin 2 (HBD2) for functional studies by the development of a method for the rapid expression and purification of the recombinant peptide. A clone encoding a thioredoxin-HBD2 fusion protein was designed for the expression of soluble peptide in E. coli cells that was purified by simple affinity chromatography. The HBD2 peptide was cleaved from the fusion by an efficient protease step and further purified to yield pure HBD2. This recombinant HBD2 defensin was shown to be active against a Mycobacterium tuberculosis mutant strain.

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.

Dissertation (DPhil)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is the only potential renewable and sustainable source of energy available to mankind at present, especially in the production of transportation fuels. Transportation fuels such as gasoline can be blended with or completely replaced by ethanol produced exclusively from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce carbon emissions and also the dependence on foreign oil (mostly from the Middle East and Africa) for many countries. Bio-ethanol can be produced from both starch and cellulose present in plants, even though cellulosic ethanol has been suggested to be the more feasible option. Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic action of acids or enzymes, which can, in turn, be broken down to monosaccharides such as hexoses and pentoses. These simple sugars can then be fermented to ethanol by microorganisms. Among the innumerable microorganisms present in nature, the yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the ‘workhorse’ of the alcohol industry. Although S. cerevisiae has arguably a relatively wide substrate utilisation range, it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have been made. Through molecular cloning methods, xylose pathway genes from the natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have been cloned and expressed separately in various S. cerevisiae strains. However, recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and fermented this pentose sugar to xylitol. The main focus of this study was to improve xylose utilisation by a recombinant S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic fermentation conditions. This has been approached at three different levels: (i) by creating constitutive carbon catabolite repression mutants in the recombinant S. cerevisiae background so that a glucose-like environment is mimicked for the yeast cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase gene from the natural xylose-degrading fungus Neurospora crassa through functional complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting mutant that showed an altered product profile after anaerobic xylose fermentation. From the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed, although ethanol formation appears to be a highly regulated process in the cell. In summary, this work exposits three different methods of improving xylose utilisation under anaerobic conditions through manipulations at the molecular level and metabolic level. The novel S. cerevisiae strains developed and described in this study show improved xylose utilisation. These strains, in turn, could be developed further to encompass other polysaccharide degradation properties to be used in the so-called consolidated bioprocess. / AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is. Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe. Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en Afrika) te verminder. Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word, maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die alkoholbedryf gemaak. Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam, Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk, het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis. Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader: (i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk te wees. Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit wat in die sogenaamde gekonsolideerde bioproses gebruik kan word.

published_or_final_version / Surgery / Master / Master of Philosophy