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Thesis (PhD) - Swinburne University of Technology, 2008. / Submitted for the degree of Doctor of Philosophy, Swinburne University of Technology - 2008. Typescript. Includes bibliographical references (p. 195-208).

The coconut rhinoceros beetle, an economically important pest of coconut and oil palms, is effectively managed by application of its natural pathogen, the Oryctes nudivirus (OrNV), which act as a bioinsecticide. While this approach offers an environment-friendly alternative to chemical pesticides, the current method of production in infected larvae suffers from inconsistencies in virus productivity and purity. While the anchorage-dependent DSIR-HA-1179 insect cell line has been identified as a susceptible and permissive host for OrNV and therefore would be suitable for the in vitro mass production of the virus, no attempts have been made toward the mass production of the virus, because of the technological challenges that working with DSIR-HA-1179 cells represent. Thus, the main objective of this research was to develop processes for the in vitro production of OrNV in the DSIR-HA-1179 cell line. Knowledge of the growth kinetics and metabolic properties of the host cell line in a chosen culture medium, as well as the selection of an appropriate infection strategy, form the basis for the rational development of bioreactor-based virus production processes. However, characterization of these properties in the DSIR-HA-1179 cell line has been virtually precluded, due to its strongly adherent growth characteristics and the lack of a reliable method to accurately dissociate and count cells grown in monolayers. Using TrypLE™ Express enzyme, a technique allowing the precise counting of cells was developed. The cell line was adapted to grow in four serum-supplemented culture media: TC-100, IPL-41, Sf-900 II and Sf-900 III, which were then individually screened for cell growth and virus production in 25 cm2 attached T-flask cultures. TC-100 supplemented with 10% fetal bovine serum was chosen as a suitable culture medium, based on its capacity for achieving a high cell yield and OrNV production. The cell line metabolism was characterized with respect to nutrient consumption and metabolites production in this culture medium. Glucose, along with glutamine were found to be the nutrients that were consumed faster and to a greater extent, while other amino acids were not consumed to a significant degree. The production of metabolites was characterized by non-production of lactate and ammonia, and production of alanine, as a non-toxic alternative to ammonia. The influence of cell density (CD) at time of infection (TOI) and multiplicity of infection (MOI) on OrNV production was evaluated in T-flask cultures that were infected at different CDs at the TOI and a range of MOIs. The CD at TOI was found to significantly influence OrNV yields, while MOI influenced the dynamics of infection. The cell density effect was found to exist for the DSIR-HA-1179/OrNV system with the progressive decline in cell-specific yield beginning at low cell densities. It was found that in order to maximize OrNV volumetric yield, a combination of MOI and CD at TOI should be selected that allows to keep the maximum cell density reached by the infected culture within a range between 5.0 and 7.0 x 105 viable cells/ml. The roller bottle system was evaluated for its potential to scale-up DSIR-HA-1179 cell growth and OrNV production, and culture parameters were optimized for the improvement of cell and virus yields. An inoculum density of 3.3 x 105 cells/ml and culture volume of 60 ml resulted in the highest cell yield of 1.5 x 106 cells/ml, in 490 cm2 roller bottles. It was found that an optimal infection strategy for roller bottle cultures, which represented the most efficient use of viral inoculum, involved infecting cells at a density of 5.0 x 105 cells/ml and at a MOI of 1. The resulting OrNV volumetric yield of 2.5 x108 TCID50/ml, improved significantly the viral yields obtained in attached T-flask cultures infected under similar conditions (6.8 x 107 TCID50/ml). The microcarrier system was also evaluated for culturing DSIR-HA-1179 cells and producing OrNV in spinner flask bioreactors. Three types of microcarriers (Cytodex-1, Cytodex-3 and Cultispher-G microcarriers) were screened for their ability to support DSIR-HA-1179 growth. Cells attached to Cytodex-1 and 3, but failed to attach to Cultispher-G microcarriers. The final cell density reached in microcarrier culture was dependent on bead type and concentration, and the cell to bead ratio. At an optimal bead concentration of 1 mg/ml and cell to bead ratio of 30, cells grew to a maximum density of 1.7 x 106 cells/ml on Cytodex-1, but only to 1.3 x 106 cells/ml on Cytodex-3 microcarriers. Since it supported higher cell yields, Cytodex-1 was chosen to study the kinetics of OrNV production in this system. Microcarrier cultures infected at a cell density of 5.0 x 105 cells/ml and a MOI of 1, produced OrNV at 1.4 x 108 TCID50/ml, which was higher than the yield obtained in T-flask cultures infected under similar conditions. A framework of knowledge on the physiology, metabolism and growth kinetics of the DSIR-HA-1179 insect cell line has been developed in this thesis. In addition, the feasibility of using roller bottles and microcarrier systems for the in vitro production of the virus has been ascertained. It is envisaged that these findings will contribute to the future development of a large-scale industrial process for the production of the OrNV biopesticide.

The existing seasonal influenza vaccine does not provide broad long-term protection against seasonal influenza and must be remanufactured yearly due to frequens mutations and reassortment of theinfluenza genes. A universal influenza vaccine with the ability to raise long lasting immunity is the focus of several studies, including the Edufluvac project. Edufluvac is based on virus-likeparticles, a modern recombinant platform wellsuited for vaccinatin applications. Redbiotec's rePAX® technology allows the generation of multivalent recombinant baculovirus which generatesvirus-like particles presenting multiple proteins on the surface in insect cell culture. Any effects oninsect cell culture protein expression brought on by the regulatory elements controlling each gene in the baculovirus, or by the genome position of the baculovirus genes, could affect the composition of the virus-like  particles. The ai of this thesis was to elicit a better understanding of the protein expression by analysing multi-protein influenza virus-like particles and virus-like particles encoding a reporter gene regulated by different promoter and terminator combinations. Different bivalent and tetravalent influenza gene bacmids were cloned as well as seven bacmids encoding a YFP gene regulated by different promoter and terminator combinations. Spodopera frugiperda cells weretransfected with the bacmids and harvested recombinant baculovirus was used to perform testexpressions in High-Five™ cells. The resulting protein expression levels from the bivalent andtetravalent recombinant baculovirus were analyzed and compared by Western blots and ELISA assays. The expression of YFP in infected Spodoptera and High-Five™ cells was monitored byfluorescence microscopy and measured with FACS to quantify protein expression differencesbetween the seven promoter and terminator combinations. Analysis of the bivalent constructs indicated that the order of the genes in a recombinant baculovirus does not affect the protein expression in High-Five™ cells. The analysis of the tetravalent constructs revelaed positionalvariations in expressin of the H1 and M1 genes, but the number of test expressions and recombinant baculovirus construct clones included in the analysis were not hogh enough to allow a definitive conclusion. Of the different promoter and terminator constructs highest mean fluorescence intensity was reached with the reference combination. The early promoter yielded mean fluorescent intensitites that were close to the values of the negative control in both cell lines.

Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula, a SEPT4 foi utilizada como isca para ensaios de interação proteína-proteína pela técnica de duplo híbrido. Para isso, o gene da SEPT4 foi clonado fusionado ao domínio de ligação ao DNA Lex-A. A realização do ensaio de duplo híbrido com a proteína completa não foi possível, pois a mesma provocou a auto ativação do sistema, por isso uma nova construção foi realizada com a região GTPase e C-terminal SEPT4GC (124-478) como isca. Dentre as interações identificadas, foram encontradas apenas septinas do grupo II (SEPT6, SEPT8, SEPT10 e SEPT11) e quatro novas interações, que ainda precisam ser confirmadas. Por outro lado, uma interação já descrita na literatura envolve a proteína &#945-sinucleína, que é uma proteína abundantemente expressa no cérebro e associada à doença de Parkinson. O foco do estudo dessa interação foi realizar ensaios com os diferentes domínios da SEPT4 para comprovar uma interação direta e com isso tentar mapear o sítio de interação com a &#945-sinucleína. Os resultados obtidos pela ressonância plasmônica de superfície (SPR) indicam que o domínio C-terminal participa da interação com baixa afinidade (K,D=390 &#181M) e sugerem que o domínio GTPase também pode estar envolvido. Já os dados obtidos com os experimentos de RMN e anisotropia de fluorescência mostram indícios que a interação é dependente da conformação da &#945-sinucleína por que a interação aconteceria com maior afinidade quando a &#945-sinucleína está na presença de SDS. / Septins are a family of GTP binding proteins found in a great diversity of organisms. These proteins have been identified as having a central role in septum organization during yeast division. Septins are organized into heterofilaments which are 7 to 9 nm wide and these have been purified from yeast, Drosophila and mice brain. Septins are not only required for cell division, but seem to play a role also in vesicle trafficking and in the formation of diffusion barriers within cells, since they interact with cytoskeleton proteins and the plasma membrane causing changes in cell morphology. In the present work, the aim was investigate human Septin 4 (SEPT4), a septin highly expressed in the brain. One objective of this work was to find a suitable expression system and purification method for SEPT4. The protein was expressed in both E.coli and insect cells (Sf-9). Expression in E. coli with the vector pETTEV was unsuccessful because the protein was insoluble. Expression in insect cells using the recombinant baculovirus AcSept4, was obtained successfully, but the purification was difficult. Important information concerning SEPT4 function might be acquired, if interactions partners involved in cellular process were identified. With this goal in mind, a yeast two hybrid assays were performed. The sept4 gene was fused to the Lex-A DNA binding domain and used as bait in the yeast two hybrid essays. However, full length SEPT4 showed autonomous activation of reporter genes. A second construct was prepared including only GTPase domain and the carboxy terminus domain, (residues 124 to 478) and the screen of interactions were carried out only with SEPT4GC. All of the group II septins (SEPT6, SEPT8, SEPT10 and SEPT11) were identified together with four new interactions. The latter still need be confirmed. In addition, another interaction already described in the literature is between SEPT4 and &#945-synuclein, which is a protein highly expressed in brain and related to Parkinson\'s disease. Different spectroscopic methods and SPR were used to identify which domain of SEPT4 interacts directly with &#945-synuclein and in which region. The surface plasmon resonance (SPR) results indicate that the carboxy terminus participates in the interaction with low affinity (KD = 390 &#181M) and suggests that the GTPase domain may also be involved. The results obtained by fluorescence anisotropy and NMR studies provide evidence that the interaction is dependent on the &#945-synuclein conformation, because the affinity of SEPT4 and &#945-synuclein seemed to be higher in the presence of SDS.

Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula, a SEPT4 foi utilizada como isca para ensaios de interação proteína-proteína pela técnica de duplo híbrido. Para isso, o gene da SEPT4 foi clonado fusionado ao domínio de ligação ao DNA Lex-A. A realização do ensaio de duplo híbrido com a proteína completa não foi possível, pois a mesma provocou a auto ativação do sistema, por isso uma nova construção foi realizada com a região GTPase e C-terminal SEPT4GC (124-478) como isca. Dentre as interações identificadas, foram encontradas apenas septinas do grupo II (SEPT6, SEPT8, SEPT10 e SEPT11) e quatro novas interações, que ainda precisam ser confirmadas. Por outro lado, uma interação já descrita na literatura envolve a proteína &#945-sinucleína, que é uma proteína abundantemente expressa no cérebro e associada à doença de Parkinson. O foco do estudo dessa interação foi realizar ensaios com os diferentes domínios da SEPT4 para comprovar uma interação direta e com isso tentar mapear o sítio de interação com a &#945-sinucleína. Os resultados obtidos pela ressonância plasmônica de superfície (SPR) indicam que o domínio C-terminal participa da interação com baixa afinidade (K,D=390 &#181M) e sugerem que o domínio GTPase também pode estar envolvido. Já os dados obtidos com os experimentos de RMN e anisotropia de fluorescência mostram indícios que a interação é dependente da conformação da &#945-sinucleína por que a interação aconteceria com maior afinidade quando a &#945-sinucleína está na presença de SDS. / Septins are a family of GTP binding proteins found in a great diversity of organisms. These proteins have been identified as having a central role in septum organization during yeast division. Septins are organized into heterofilaments which are 7 to 9 nm wide and these have been purified from yeast, Drosophila and mice brain. Septins are not only required for cell division, but seem to play a role also in vesicle trafficking and in the formation of diffusion barriers within cells, since they interact with cytoskeleton proteins and the plasma membrane causing changes in cell morphology. In the present work, the aim was investigate human Septin 4 (SEPT4), a septin highly expressed in the brain. One objective of this work was to find a suitable expression system and purification method for SEPT4. The protein was expressed in both E.coli and insect cells (Sf-9). Expression in E. coli with the vector pETTEV was unsuccessful because the protein was insoluble. Expression in insect cells using the recombinant baculovirus AcSept4, was obtained successfully, but the purification was difficult. Important information concerning SEPT4 function might be acquired, if interactions partners involved in cellular process were identified. With this goal in mind, a yeast two hybrid assays were performed. The sept4 gene was fused to the Lex-A DNA binding domain and used as bait in the yeast two hybrid essays. However, full length SEPT4 showed autonomous activation of reporter genes. A second construct was prepared including only GTPase domain and the carboxy terminus domain, (residues 124 to 478) and the screen of interactions were carried out only with SEPT4GC. All of the group II septins (SEPT6, SEPT8, SEPT10 and SEPT11) were identified together with four new interactions. The latter still need be confirmed. In addition, another interaction already described in the literature is between SEPT4 and &#945-synuclein, which is a protein highly expressed in brain and related to Parkinson\'s disease. Different spectroscopic methods and SPR were used to identify which domain of SEPT4 interacts directly with &#945-synuclein and in which region. The surface plasmon resonance (SPR) results indicate that the carboxy terminus participates in the interaction with low affinity (KD = 390 &#181M) and suggests that the GTPase domain may also be involved. The results obtained by fluorescence anisotropy and NMR studies provide evidence that the interaction is dependent on the &#945-synuclein conformation, because the affinity of SEPT4 and &#945-synuclein seemed to be higher in the presence of SDS.

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

O cDNA da glicoproteína do vírus da raiva (GPV) foi clonado em vetores plasmidiais (indutíveis) contendo ou não o cDNA do sinal de secreção BiP e da resistência ao antibiótico higromicina B. Esses vetores foram transfectados em células S2 e foram obtidas populações e subpopulações. A população S2MTGPV-H apresentou níveis 5x maiores na expressão da GPV em análise por FACS (~ 50% das células) e por ELISA (~ 0,65 µg/107 células). A seleção de subpopulações permitiu um aumento de aproximadamente 10x na expressão da GPV, especialmente na população S2MTGPV*-H. O tratamento com NaBu resultou em uma redução de aproximadamente 20% no crescimento celular e um aumento de 50% na GPV expressa pela população S2MTGPV*-H (~ 8,3 µg/107 células). O meio de cultura SF900 II permitiu um maior crescimento das células S2MTGPV*-H e uma maior síntese de GPV comparado com outros meios de cultura. Nossos dados mostram que a expressão da GPV pôde ser otimizada através da construção de vetores de expressão/seleção, subpopulações, da exposição da cromatina e do meio de cultura utilizado. / The cDNA encoding the entire rabies virus glycoprotein (RVGP) gene was cloned in plasmids (inductive) with or without a cDNA coding for the secretion signal and coding for the selection hygromicin antibiotic. These vectors were transfected into S2 cells and we had obtain cells populations and subpopulations S2MTRVGP-H cell population were shown to express 5 times higher of RVGP as evaluated by FACS (~ 50 %) and ELISA (~ 0.65 mg/107 cells at day 7). Sub-population selection allowed a higher RVGP expression, especially for the S2MTRVGP*-H. NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MTRVGP*-H (~ 8.3 mg/107 cells at day 7 after induction). SF900II medium leading to a higher S2MTRVGP*-H cell growth allowed a higher final RVGP synthesis in this cell culture. The data show that RVGP synthesis may be optimized by the expression/selection vectors design, cell sub-populations selection, chromatine exposure and culture medium employed.

O interesse em células de inseto para a produção de proteínas complexas se deve a sua maior facilidade de cultivo e ao padrão equivalente de glicosilação quando comparado aos sistemas com células de mamíferos. O objetivo deste trabalho foi identificar fatores que limitam ou inibem a produção da glicoproteína do vírus rábico (GPV) expressa na membrana citoplasmática de células de Drosophila melanogaster transfectadas, quando cultivadas em biorreator de bancada agitado e bubble-free, operado em modo descontínuo. Avaliaram-se as influências de oxigênio dissolvido (5 &lt; pO2 &lt;80%), da glicose (1 &lt; GLC0 &lt; 15g/L) e da glutamina (0.6 &lt; GLN0 &lt; 7g/L). Essas variáveis afetaram de forma diferenciada o crescimento celular (produção de células e velocidades específicas-&#181;X), o metabolismo celular (fatores de conversão - YX/GLC, YX/GLN, YLAC/GLC, YALA/GLC, YNH4/GLN, YALA/GLN), assim como a expressão da proteína recombinante (concentração, teor celular e produtividade). O aumento do pO2 reduziu em 9 vezes o crescimento celular mas aumentou o teor celular de GPV em 1,4 vezes. Baixos valores de GLC0 e GLN0, claramente, limitaram o crescimento, de modo que incrementos na concentração desses substratos, até valores intermediários, aumentaram &#181;X,MAX em 3 vezes e 2,5 vezes, respectivamente, e a produção de células em 11 vezes e 3 vezes, respectivamente. O teor celular de GPV máximo não foi afetado pela GLC, mas aumentou em 100% para valores de GLN0 igual ou superiores a 3,5 g/L. As concentrações de lactato produzidas foram consideradas baixas (inferiores a 0,8 g/L) para exercer qualquer efeito de inibição sobre o crescimento ou a expressão da proteína. Por sua vez, as concentrações de amônio parecem inibir tanto a produção de GPV (NH4+~50mg/L) quanto o crescimento celular (NH4+~80mg/L). A condição de cultivo com de 30% de pO2, 10 g/L de GLC0 e 3,5 g/L de GLN0 resultou nos maiores valores de produtividade (9,1 &#181;g/L.h) e de concentração de GPV (1,2 mg/L). O metabolismo de GLC e GLN apresentou grande interdependência, com alterações em GLC0 afetando o metabolismo de GLN e vice-versa. Assim, em condições de excesso de GLC0, as células apresentaram um metabolismo mais ineficiente com reduções nos fatores YX/GLC (2,3 vezes) e YX/GLN (4,6 vezes) e maior geração de subprodutos, caracterizada por incrementos nos valores de YALA/GLC (51%), YLAC/GLC (11%) e YNH4/GLN (15%). O metabolismo da GLN apresentou resposta característica de substrato em excesso para toda a faixa de valores ensaiada, com redução de 25 vezes no valor de YX/GLN e inesperadamente também uma redução na geração de subprodutos de 7 vezes para YNH4/GLN e 12 vezes para YALA/GLN. O efeito sobre o metabolismo da GLC foi mais acentuado para valores mais elevados de GLN0, com redução de 3,6 vezes para YX/GLC e incrementos de 70% para YALA/GLC e para YLAC/GLC. Os resultados sugerem ainda que a célula utiliza duas vias para metabolizar a glutamina: glutaminólise, em condição de limitação em GLC; ou glutamato sintase - NADH-GOGAT, em condição de excesso em GLC. A célula demonstrou também capacidade de sintetizar GLN, a partir de amônio ou outros aminoácidos, quando atingiu concentrações abaixo de 50 mg/L. / The interest in using insect cells to produce complex proteins is due to its ease of cultivation and its glycosylation pattern equivalent to that of mammalian cells systems. The objective of this work was to identify the limiting or inhibiting factors for the production of a rabies virus glycoprotein (RVGP), expressed in the cytoplasmatic membrane of a transfected Drosophila melanogaster S2 cells, when cultivated in a bench stirred bubble-free bioreactor, in batch mode. The influence of dissolved oxygen (5 &lt; pO2 &lt; 80%), of initial glucose concentration (1 &lt; GLC0 &lt; 15 g/L) and of initial glutamine concentration (0.6 &lt; GLN0 &lt; 7 g/L) was evaluated. These variables affected in a different way cell growth (cell production and specific growth rate - &#181;X), cell metabolism (yield factors - YX/GLC, YX/GLN, YLAC/GLC, YALA/GLC, YNH4/GLN and YALA/GLN), as well as the recombinant protein expression (RVGP concentration, RVGP cell content and RVGP productivity). pO2 increase reduced 9 times cell growth, but increased 1.4 times RVGP cell content. Low initial glucose and glutamine concentrations clearly limited the cell growth, in such a way that raising these substrates concentrations up to intermediate values, increased &#181;X,MAX 3 times and 2.5 times, respectively, and increased cell production 11 times and 3 times, respectively. The maximum RVGP cell content was not affected by GLC0, but improved 100% when GLN0 was 3.5 g/L or higher. The concentrations of produced lactate were considered low (below 0.8 g/L) to cause any inhibition effect on growth or protein expression. On the other hand, ammonium concentrations seem to inhibit RVGP production (NH4+~50 mg/L), as well as cell growth (NH4+~80 mg/L). Maximum productivity values (9.1 &#181;g/L.h) and RVGP concentration (1.2 mg/L) were attained for 30% pO2, 10 g/L of GLC0 and 3.5 g/L of GLN0 run. The metabolism of GLC and GLN showed a great interdependence, with GLC0 changes affecting the GLN metabolism, and viceversa. Thus, in glucose excess condition, cell metabolism was less efficient. This implied in reduction of yield factors - YX/GLC (2.3 times) e YX/GLN (4.6 times) - and in higher by-products generation, characterized by augmentation in YALA/GLC (51%), YLAC/GLC (11%) and YNH4/GLN (15%). The glutamine metabolism showed a substrate excess response pattern to the whole range of concentration studied, with reduction of YX/GLN (25 times) and, unexpectedly, a reduction of by-products liberation - YNH4/GLN (7 times) and YALA/GLN (12 times). The effect on glucose metabolism was more intense when the glutamine concentration was higher, showing a 3.6 times diminution YX/GLC and a 70% augmentation for YALA/GLC and YLAC/GLC. The results suggest that cells metabolize glutamine through two different pathways glutaminolysis, under glucose limitation, or glutamate synthase - NADH-GOGAT, under glucose excess. The cell, proved also to be able to synthesize glutamine from ammonium or other amino acids, when it reached concentrations below 50 mg/L.

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.