Superprodu??o do interferon β1 humano recombinante em escherichia coli

Made available in DSpace on 2015-04-14T14:50:48Z (GMT). No. of bitstreams: 1 400016.pdf: 696310 bytes, checksum: 7f8908ad96586598d5473332eb4bd96b (MD5) Previous issue date: 2008-03-26Interferons s?o prote?nas expressas por diferentes c?lulas humanas e sintetizadas como resposta a muitos agentes qu?micos e biol?gicos. Eles est?o envolvidos em repostas antiviral, antiproliferativa e imunomodulat?ria, atuando na manuten??o da homeostase e na prote??o do organismo contra pat?genos. Devido a essa atividade biol?gica, os interferons s?o atualmente aprovados no mundo inteiro para o tratamento de v?rias desordens virais, malignas e imunol?gicas. Interferon β1, por exemplo, ? uma das poucas subst?ncias que provou ser efetiva na supress?o das manifesta??es da esclerose m?ltipla que ? uma doen?a cr?nica do sistema nervoso central. Ele ? produzido comercialmente em microorganismos como Escherichia coli, utilizando a tecnologia do DNA recombinante e ? chamado de biof?rmaco. As patentes de muitos biof?rmacos originais est?o expirando e uma nova gera??o de mol?culas, chamadas biossimilares, est? em desenvolvimento. O interferon β1 foi aprovado para uso no tratamento da esclerose m?ltipla surtoremiss?o pelo Departamento de Controle de Drogas e Alimentos dos EUA em 1993 e perdeu sua prote??o de patente em 2007, tornando-se um alvo para a produ??o do biossimilar pelas ind?strias farmac?uticas. O desenvolvimento do biossimilar interferon β1 ? uma alternativa para o alto custo do biof?rmaco original. Desenvolvemos um protocolo para produzir grandes quantidades do interferon β1, no qual aproximadamente 3 mg da prote?na recombinante homog?nea podem ser obtidos a partir de 1 g de c?lulas de E. coli Rosetta(DE3). As an?lises por seq?enciamento N-terminal de amino?cidos e espectrometria de massas proveram evid?ncias para a identidade e pureza da prote?na recombinante. A an?lise por cromatografia l?quida de fase reversa demonstrou que o conte?do de deamidados e sulf?xidos foi similar ao padr?o comercial, e nenhuma forma heterog?nea da prote?na rhIFN-β1ser17 foi detectada. A an?lise por cromatografia l?quida de exclus?o molecular demonstrou a aus?ncia de agregados de alta massa molecular e de d?meros. O protocolo desenvolvido poder? ser utilizado para a produ??o do biossimilar pelas ind?strias farmac?uticas e deste modo, diminuir os custos do Minist?rio da Sa?de e dos consumidores com este biof?rmaco

Estudos bioqu?micos e nocaute g?nico da enzima uracil fosforribosil transferase de Mycobacterium tuberculosis como alvo para o desenvolvimento de cepas atenuadas

Made available in DSpace on 2015-04-14T14:51:16Z (GMT). No. of bitstreams: 1 437675.pdf: 3014292 bytes, checksum: 10c5c967edfd4e61677f7867c8186a73 (MD5) Previous issue date: 2011-12-09Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis, which currently infects one-third of the world s population. Despite the availability of the Bacille Calmette-Gu?rin vaccine and effective short-course chemotherapy, the increasing global burden of TB has been linked to the co-infection with HIV, the emergence of multi, extensively and now totally drug-resistant strains. Furthermore, the capacity of M. tuberculosis to remain viable within infected hosts in a long-term asymptomatic infection is an additional problem for the control of TB. Nucleotides biosynthesis pathways provide promising molecular targets for the development of new vaccines and therapeutic strategies to control the global incidence of TB. Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5'-phosphoribosyl-a- 1'-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since its product (UMP) is a common precursor of all pyrimidine nucleotides. This work presents cloning, recombinant expression in Escherichia coli, and purification of the upp-encoded M. tuberculosis UPRT (MtUPRT). Mass spectrometry analysis and N-terminal amino acid sequencing confirmed the identity of homogeneous MtUPRT. The molecular mass of the native MtUPRT was shown to follow a monomer-tetramer association model by analytical ultracentrifugation. This enzyme did not show a pronounced regulation by GTP as this nucleotide did not affect enzyme kinetic parameters, and its binding was not detected by isothermal titration calorimetry (ITC). Initial velocity and ITC studies suggested that catalysis proceeds through a sequential ordered mechanism, in which PRPP binds first, followed by uracil binding, and PPi is the first product to be released, followed by UMP. ITC also showed that PRPP and UMP binding are thermodynamically favorable processes. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalytic activity and a group with a pK value of 9.45 is involved in PRPP binding. Pre-steady-state kinetic data suggested that product release is not the rate-limiting step of the reaction catalyzed by MtUPRT. Kinetic fluorescence studies demonstrated two forms of enzyme in solution of which only one can bind to PRPP. Knockout of the upp gene showed that this gene is not essential for M. tuberculosis H37Rv in the employed experimental conditions and the absence of upp gene did not affect the mycobacterium growth. UPRT is expressed in both high and low oxygen conditions of M. tuberculosis H37Ra growth. MtUPRT is inhibited by an active metabolite of isoxyl, which does not seem to inhibit RNA polymerase, adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase. Minimum inhibitory concentration of isoxyl for M. tuberculosis mutant for upp gene, complemented and wild type strains was 12.8 μg/mL, meaning that the absence of the upp gene did not affect M. tuberculosis sensitivity to isoxyl. Altogether, these data may be useful for a better understanding about the nucleotide biosynthesis pathway in M. tuberculosis and as a framework on which to base efforts towards the development of efficient prophylactic and therapeutic strategies to decrease the global incidence of this pathogen.A tuberculose (TB) ? uma doen?a infecciosa causada principalmente pelo Mycobacterium tuberculosis, o qual atualmente infecta um ter?o da popula??o mundial. Apesar da disponibilidade da vacina Bacille Calmette-Gu?rin e da eficaz quimioterapia de curta dura??o, o aumento na incid?ncia global da TB est? relacionado ? co-infec??o com o HIV e ao surgimento de cepas multi, extensivamente e agora totalmente resistente a drogas. Al?m disto, a capacidade do M. tuberculosis de permanecer vi?vel dentro do hospedeiro infectado por longo per?odo em uma infec??o assintom?tica ? um problema adicional para o controle da TB. As rotas de bioss?ntese de nucleot?deos fornecem alvos moleculares promissores para o desenvolvimento de novas vacinas e estrat?gias terap?uticas para controlar a incid?ncia global de TB no mundo. A uracil fosforribosil transferase (UPRT) catalisa a convers?o de uracil e 5 - fosforribosil-a-1 -pirofosfato (PRPP) a uridina 5 -monofosfato (UMP) e pirofosfato (PPi). A UPRT tem um papel importante na rota de salvamento das pirimidinas j? que seu produto (UMP) ? o precursor comum de todos os nucleot?deos pirim?dicos. Este trabalho apresenta a clonagem, express?o recombinante em E. coli, e a purifica??o da UPRT de M. tuberculosis codificada pelo gene upp (MtUPRT). Adicionalmente, foram realizadas an?lises de espectrometria de massas e sequenciamento N-terminal que confirmaram a identidade da MtUPRT homog?nea. A massa molecular da MtUPRT nativa seguiu um modelo de associa??o mon?mero-tetr?mero por ultracentrifuga??o anal?tica. Esta enzima n?o mostrou uma regula??o pronunciada por GTP j? que este nucleot?deo n?o afetou os par?metros cin?ticos da enzima, e a sua liga??o n?o foi detectada por calorimetria de titula??o isot?rmica (ITC). A velocidade inicial e os estudos de ITC sugeriram que a cat?lise procede atrav?s de um mecanismo ordenado sequencial, no qual o PRPP liga primeiramente, seguido pela liga??o de uracil, e PPi ? o primeiro produto a ser liberado, seguido pelo UMP. ITC tamb?m mostrou que a liga??o de PRPP e UMP s?o processos termodinamicamente favor?veis. O perfil de pH indicou que grupamentos com os valores de pK pr?ximos de 5,7 e 8,1 s?o importantes para a atividade catal?tica e um grupamento com valor de pK pr?ximo a 9,45 est? envolvido na liga??o do PRPP. Dados de cin?tica no estado pr?-estacion?rio sugeriram que a libera??o de produto n?o ? a etapa limitante da rea??o catalisada pela MtUPRT. Estudos de fluoresc?ncia demonstraram a exist?ncia de duas formas da enzima em solu??o, das quais apenas uma pode ligar ao PRPP. O nocaute do gene upp demonstrou que este gene n?o ? essencial para o M. tuberculosis H37Rv nas condi??es empregadas no experimento e a aus?ncia do gene upp n?o afetou o crescimento micobacteriano. A UPRT mostrou ser expressa tanto em altas como em baixas concentra??es de oxig?nio. A MtUPRT foi inibida por um metab?lito ativo do isoxyl, que n?o parece inibir as enzimas RNA polimerase, adenina fosforribosil transferase e hipoxantinaguanina fosforribosil transferase. A concentra??o inibit?ria m?nima de isoxyl para as cepas de M. tuberculosis mutante para o gene upp, complementada e tipo selvagem foi de 12,8 μg/mL, o que significa que a aus?ncia do gene upp n?o afetou a sensibilidade do M. tuberculosis ao isoxyl. Assim, estes dados podem ser ?teis para um melhor entendimento sobre a via de bioss?ntese de nucleot?deos em M. tuberculosis e podem servir como base para o desenvolvimento de estrat?gias terap?uticas e preventivas eficientes para diminuir a incid?ncia global deste pat?geno

Estudos bioqu?micos e nocaute g?nico da enzima uracil fosforribosil transferase de Mycobacterium tuberculosis como alvo para o desenvolvimento de cepas atenuadas

Made available in DSpace on 2015-04-14T14:51:16Z (GMT). No. of bitstreams: 1 437675.pdf: 3014292 bytes, checksum: 10c5c967edfd4e61677f7867c8186a73 (MD5) Previous issue date: 2011-12-09Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis, which currently infects one-third of the world s population. Despite the availability of the Bacille Calmette-Gu?rin vaccine and effective short-course chemotherapy, the increasing global burden of TB has been linked to the co-infection with HIV, the emergence of multi, extensively and now totally drug-resistant strains. Furthermore, the capacity of M. tuberculosis to remain viable within infected hosts in a long-term asymptomatic infection is an additional problem for the control of TB. Nucleotides biosynthesis pathways provide promising molecular targets for the development of new vaccines and therapeutic strategies to control the global incidence of TB. Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5'-phosphoribosyl-a- 1'-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since its product (UMP) is a common precursor of all pyrimidine nucleotides. This work presents cloning, recombinant expression in Escherichia coli, and purification of the upp-encoded M. tuberculosis UPRT (MtUPRT). Mass spectrometry analysis and N-terminal amino acid sequencing confirmed the identity of homogeneous MtUPRT. The molecular mass of the native MtUPRT was shown to follow a monomer-tetramer association model by analytical ultracentrifugation. This enzyme did not show a pronounced regulation by GTP as this nucleotide did not affect enzyme kinetic parameters, and its binding was not detected by isothermal titration calorimetry (ITC). Initial velocity and ITC studies suggested that catalysis proceeds through a sequential ordered mechanism, in which PRPP binds first, followed by uracil binding, and PPi is the first product to be released, followed by UMP. ITC also showed that PRPP and UMP binding are thermodynamically favorable processes. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalytic activity and a group with a pK value of 9.45 is involved in PRPP binding. Pre-steady-state kinetic data suggested that product release is not the rate-limiting step of the reaction catalyzed by MtUPRT. Kinetic fluorescence studies demonstrated two forms of enzyme in solution of which only one can bind to PRPP. Knockout of the upp gene showed that this gene is not essential for M. tuberculosis H37Rv in the employed experimental conditions and the absence of upp gene did not affect the mycobacterium growth. UPRT is expressed in both high and low oxygen conditions of M. tuberculosis H37Ra growth. MtUPRT is inhibited by an active metabolite of isoxyl, which does not seem to inhibit RNA polymerase, adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase. Minimum inhibitory concentration of isoxyl for M. tuberculosis mutant for upp gene, complemented and wild type strains was 12.8 μg/mL, meaning that the absence of the upp gene did not affect M. tuberculosis sensitivity to isoxyl. Altogether, these data may be useful for a better understanding about the nucleotide biosynthesis pathway in M. tuberculosis and as a framework on which to base efforts towards the development of efficient prophylactic and therapeutic strategies to decrease the global incidence of this pathogen.A tuberculose (TB) ? uma doen?a infecciosa causada principalmente pelo Mycobacterium tuberculosis, o qual atualmente infecta um ter?o da popula??o mundial. Apesar da disponibilidade da vacina Bacille Calmette-Gu?rin e da eficaz quimioterapia de curta dura??o, o aumento na incid?ncia global da TB est? relacionado ? co-infec??o com o HIV e ao surgimento de cepas multi, extensivamente e agora totalmente resistente a drogas. Al?m disto, a capacidade do M. tuberculosis de permanecer vi?vel dentro do hospedeiro infectado por longo per?odo em uma infec??o assintom?tica ? um problema adicional para o controle da TB. As rotas de bioss?ntese de nucleot?deos fornecem alvos moleculares promissores para o desenvolvimento de novas vacinas e estrat?gias terap?uticas para controlar a incid?ncia global de TB no mundo. A uracil fosforribosil transferase (UPRT) catalisa a convers?o de uracil e 5 - fosforribosil-a-1 -pirofosfato (PRPP) a uridina 5 -monofosfato (UMP) e pirofosfato (PPi). A UPRT tem um papel importante na rota de salvamento das pirimidinas j? que seu produto (UMP) ? o precursor comum de todos os nucleot?deos pirim?dicos. Este trabalho apresenta a clonagem, express?o recombinante em E. coli, e a purifica??o da UPRT de M. tuberculosis codificada pelo gene upp (MtUPRT). Adicionalmente, foram realizadas an?lises de espectrometria de massas e sequenciamento N-terminal que confirmaram a identidade da MtUPRT homog?nea. A massa molecular da MtUPRT nativa seguiu um modelo de associa??o mon?mero-tetr?mero por ultracentrifuga??o anal?tica. Esta enzima n?o mostrou uma regula??o pronunciada por GTP j? que este nucleot?deo n?o afetou os par?metros cin?ticos da enzima, e a sua liga??o n?o foi detectada por calorimetria de titula??o isot?rmica (ITC). A velocidade inicial e os estudos de ITC sugeriram que a cat?lise procede atrav?s de um mecanismo ordenado sequencial, no qual o PRPP liga primeiramente, seguido pela liga??o de uracil, e PPi ? o primeiro produto a ser liberado, seguido pelo UMP. ITC tamb?m mostrou que a liga??o de PRPP e UMP s?o processos termodinamicamente favor?veis. O perfil de pH indicou que grupamentos com os valores de pK pr?ximos de 5,7 e 8,1 s?o importantes para a atividade catal?tica e um grupamento com valor de pK pr?ximo a 9,45 est? envolvido na liga??o do PRPP. Dados de cin?tica no estado pr?-estacion?rio sugeriram que a libera??o de produto n?o ? a etapa limitante da rea??o catalisada pela MtUPRT. Estudos de fluoresc?ncia demonstraram a exist?ncia de duas formas da enzima em solu??o, das quais apenas uma pode ligar ao PRPP. O nocaute do gene upp demonstrou que este gene n?o ? essencial para o M. tuberculosis H37Rv nas condi??es empregadas no experimento e a aus?ncia do gene upp n?o afetou o crescimento micobacteriano. A UPRT mostrou ser expressa tanto em altas como em baixas concentra??es de oxig?nio. A MtUPRT foi inibida por um metab?lito ativo do isoxyl, que n?o parece inibir as enzimas RNA polimerase, adenina fosforribosil transferase e hipoxantinaguanina fosforribosil transferase. A concentra??o inibit?ria m?nima de isoxyl para as cepas de M. tuberculosis mutante para o gene upp, complementada e tipo selvagem foi de 12,8 μg/mL, o que significa que a aus?ncia do gene upp n?o afetou a sensibilidade do M. tuberculosis ao isoxyl. Assim, estes dados podem ser ?teis para um melhor entendimento sobre a via de bioss?ntese de nucleot?deos em M. tuberculosis e podem servir como base para o desenvolvimento de estrat?gias terap?uticas e preventivas eficientes para diminuir a incid?ncia global deste pat?geno

Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP(i) product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development