Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni²⁺ Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min⁻¹ was obtained whereas for ADP release a greater kcat value of 0.8 min⁻¹ was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.

Orientador: Anete Pereira de Souza / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T10:23:34Z (GMT). No. of bitstreams: 1 Soares_JoseSergiodeMacedo_M.pdf: 3092877 bytes, checksum: 85276ce21b221869d2fb74dc6cef8444 (MD5) Previous issue date: 2007 / Resumo: O fitopatogeno Xylella fastidiosa é uma bactéria limitada ao xilema que causa uma variedade de doenças economicamente importantes em plantas, incluindo uma doença chamada Clorose Variegada de Citrus (CVC). Recentemente, os dados de sequenciamento do DNA fornecidos pelo projeto genoma da Xylella fastidiosa permitiram uma aproximação da área genoma funcional para investigar a função de diversas proteínas baseadas na informação sobre suas estruturas. Avanços recentes na área genoma estrutural não somente nos ajudou a compreender funções da proteína, mas também causou um grande impacto na indústria farmacêutica. Nos últimos anos, o uso da informação estrutural de proteínas na pesquisa de descoberta de novas drogas tem amadurecido, e é usado agora em todos os níveis, variando da identificação e da seleção de alvos do genoma a candidatos apropriados. Esta emergente área é poderoso meio para compreender mais profundamente os mecanismos a patogenicidade da bactéria. Este trabalho visa adicionar novas informações sobre proteínas que podem ser relacionadas a patogenicidade de Xylella fastidiosa, necessárias para o desenvolvimento de novos métodos de combate à CVC. A fim de recolher informações sobre as proteínas envolvidas nos mecanismos do patogenicidade da bactéria, nós escolhemos as orf xf2148 e orf xf1524 de Xylella fastidiosa para estudos de caracterização. A orf xf2148, apresenta 47% de similaridade com o gene ksgA de Escherichia coli. O gene ksgA codifica uma Dimetiladenosina transferase da classe de proteínas metiltransferases. Já a orf xf1524, apresenta 55% de similaridade com o gene xpsL de Xanthomonas campestris, que codifica uma proteína transmembrana da via de secreção do tipo II. Ambas as proteínas foram clonadas no vetor pET32Xa/LIC e expressas na bactéria Escherichia coli linhagem BL21(DE3). A proteína XPSL foi purificada através de uma cromatografia de afinidade (IMAC) e teve sua identidade determinada por SDS-PAGE e Espectrometria de Massas (MALDITOF). Para investigar a integridade estrutural da proteína XPSL purificada, a técnica espectroscópica de Dicroísmo Circular (CD) foi realizada. O espectro de CD da proteína XPSL mostrou sinais predominantemente de 'alfa'hélices indicando um perfil de estrutura secundária devidamente enovelada viável para estudos estruturais e funcionais. A proteína XF2148, expressa em sua forma insolúvel, foi resolubilizada usando 8M de uréia como agente desnaturante. A proteína foi purificada através de uma cromatografia de troca iônica e sua pureza e identidade verificadas por SDS-PAGE e Espectrometria de Massas (MALDI-TOF). Seu correto enovelamento por espectroscopia de Dicroísmo Circular (CD) indicou uma composição predominantemente de 'alfa'hélices. O alinhamento da seqüência primária e modelagem por homologia da proteína XF2148 com uma proteína de estrutura tridimensional resolvida, KsgA de E. coli, revelaram um local da molécula que envolve resíduos altamente conservados no domínio C-terminal e cinco dos oito motivos estruturais encontrados geralmente em Metiltransferases / Abstract: The phytopathogen Xylella fastidiosa is a xylem-limited bacterium that causes a range of economically important plant diseases, including a serious disease of orange trees called citrus variegated chlorosis (CVC). Recently, DNA sequence data provided by the Xylella fastidiosa Brazilian genome project have allowed a functional genomic approach to investigate the function of several proteins based on the information about their structures. Recent advances in structural genomics not only help us to understand protein functions but also have a big impact on the pharmaceutical industry. In the last few years, the use of protein structural information in drug discovery research has matured, and it is now used at all levels, ranging from genomics target identification and selection to the final design of suitable drug candidates. This emergent area is a powerful means to more deeply understand the mechanisms of the bacterium pathogenicity. This work aims at adding new information on proteins that may be related to the X. fastidiosa pathogenesis, necessary for new approaches towards the combat of CVC. In order to gather information about the proteins involved in the mechanisms of the bacterium pathogenicity, we chose orf xf2148 and orf xf1524 from Xylella fastidiosa for characterization studies. Orf xf2148, presents 47% of similarity with the gene ksgA of Escherichia coli. The ksgA gene encodes a dimethyladenosine transferase from the methyl transferase proteins class. On the other hand orf xf1524, presents 55% of similarity with the xpsL gene of Xanthomonas campestris, which encodes a transmembrane protein of the type II secretion machinary. Both proteins were cloned into the pET32Xa/LIC vector to overexpress the protein in Escherichia coli BL21(DE3). The expressed XpsL protein was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by SDS-PAGE and Mass Spectrometry (MALDI-TOF). To investigate the structural integrity of the purified XPSL, the protein was analyzed by Circular Dichroism (CD) spectroscopy. The XpsL CD spectrum showed that it contains predominantly signal of 'alfa'helices suggesting that the XpsL recombinant protein maintains secondary structures, is viable to functional and structural studies and remained folded. The XF2148 protein was expressed in the insoluble form and refolded using 8M Urea as denaturating agent. The protein was purified in one step by cation-exchange chromatography and its purity and identity were verified by SDS-PAGE and Mass Spectrometry (MALDI-TOF). Its correct folding was verified by Circular Dichroism Spectroscopy analysis that indicated a secondary structure composed mainly of 'alfa'helices. The alignment of the XF2148 primary sequence and homology modeling with one ksgA structure solved protein from E. coli, revealed a site involving highly conserved residues in the C-terminal domain and five of the eight structural motifs usually found in AdoMetdependent methyltransferases / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Orientador: Hiroshi Aoyama / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T06:33:37Z (GMT). No. of bitstreams: 1 Sousa_RobertaReginaRuelade_M.pdf: 3257089 bytes, checksum: 29f24a432f8c0bfd7dd71af48cc7608a (MD5) Previous issue date: 2005 / Resumo: As proteínas tirosina fosfatases (PTP) (EC 3.1.3.48) são enzimas regulatórias chaves que participam na transdução de sinal e são essenciais na regulação do crescimento, diferenciação, ciclos celulares, na transcrição gênica, resposta imune e outros processos. Esta classe de enzimas, que contém cisteína no sítio ativo, pode ser inativada por agentes oxidantes. Neste trabalho, estudamos os efeitos de peróxido de hidrogênio e t-butil hidroperóxido, compostos que induzem estresse oxidativo, na atividade de uma PTP purificada de membranas de linfócitos humanos, indicativamente a CD45. A PTP foi purificada de membranas de linfócitos humanos através de cromatografias de troca iônica (DEAE Sepharose) e exclusão molecular (Sephacryl S-200). A purificação enzimática foi acompanhada por SDS-PAGE e eletroforese bidimensional. A atividade enzimática foi determinada através de incubação a 37°C por 30 min em pH 5,0 em presença de 5 mM de p-nitrofenil fosfato (pNPP) como substrato. A enzima obtida da cromatografia de exclusão molecular apresentou uma massa molecular relativa de aproximadamente 200 kDa, reconheceu mais eficientemente tirosina fosfato (cerca de 3,2 vezes) como substrato quando comparado ao pNPP, e foi inibida por inibidores específicos de PTP, tais como vanadato (40%), pervanadato (100%), p-cloromercuribenzoato (20%) and Cu2+ (95%). Ácido okadáico, um inibidor específico de serina e treonina proteína fosfatases, não afetou a atividade da PTP de membranas de linfócitos. Estes resultados de caracterização sugerem fortemente que a PTP purificada de membranas de linfócitos humanos é a CD45. Peróxido de hidrogênio e t-butil hidroperóxido inibiram a atividade dessa proteína com valores de IC50 (concentração do composto que produz 50% de inibição enzimática) de 50 µM e 16 mM, respectivamente. Glutationa reduzida (GSH) protegeu parcialmente a enzima contra estes oxidantes, porém proteções totais foram obtidas quando se adicionava 250 mM de desferoxamina ao meio de ensaio. Nossos resultados sugerem que essa proteína pode ser regulada por alteração do estado de oxidação dos grupos funcionais do sítio ativo e que esta regulação poderia fornecer um mecanismo de controle celular através de espécies reativas de oxigênio / Abstract: Protein phosphatases, that dephosphorylate proteins in residues of threonine, serine and tyrosine, are a class of enzymes that can be stressed by compounds present in oxidant or reductant environments. In particular, the protein tyrosine phosphatases (PTP) (EC 3.1.3.48) are key regulatory enzymes that participate in signal transduction and are essential for regulating cellular growth, differentiation, cell cycle, gene transcription, immune response and other processes. This class of enzymes, which contain cysteine in the active site, can be inactivated by oxidant reagents. In this work we have studied the effect of hydrogen peroxide and t-butyl hydroperoxide, compounds that induce oxidative stress, on a purified PTP (CD45) from membrane human lymphocytes. PTP was purified from human lymphocyte membranes through ion exchange (DEAE Sepharose) and molecular exclusion (Sephacryl S-200) chromatographies. The enzyme purification was followed by SDS-PAGE and 2D electrophoresis. The enzyme activity was determined by incubation at 37oC for 30 minutes at pH 5.0 in presence of 5 mM p-nitrophenylphosphate (pNPP) as substrate. The enzyme obtained from molecular exclusion chromatography had a relative molecular mass of approximately 200 kDa, recognized more efficiently tyrosine phosphate (about 3.2-fold) as substrate when compared with p-NPP, and was inhibited by specific PTP inhibitors, such as, vanadate (40%), pervanadate (100%), p-chloromercuribenzoate (20%) and Cu2+ (95%). Okadaic acid, a specific serine and threonine protein phosphatases inhibitor, did not significantly affect the lymphocyte membrane PTP activity. These characterization results strongly suggest that the membrane PTP purified from human lymphocytes was the CD45. Hydrogen peroxide and t-butyl hydroperoxide inhibited the CD45 activities with IC50 (concentration of compound that produces 50% enzyme inhibition) values of 50 µM and 16 mM, respectively. Reduced glutathione (GSH) partially protected the enzyme against these oxidations, but full protections were observed when 250 mM deferoxamine were added to the assay medium. Our results suggest that CD45 can be regulated by altering the oxidation state of active site functional groups, and that this regulation could provide a mechanism of cell control by reactive oxygen species / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T09:14:39Z (GMT). No. of bitstreams: 1 Mourao_CeciliaAlves_M.pdf: 2559584 bytes, checksum: efe6b17b9b7e5ceb16ade74a6ae30629 (MD5) Previous issue date: 2014 / Resumo: As imunoglobulinas G (IgG) e seus fragmentos Fab, F(ab)¿2 e Fv apresentam aplicações proeminentes nas áreas terapêuticas e de diagnósticos. Em determinadas situações em que a região Fc é dispensável e/ou deletéria, emprega-se preferencialmente fragmentos em relação à IgG não clivada. Tais aplicações requerem um elevado grau de pureza dessas biomoléculas. O elevado custo de obtenção de fragmentos pelas técnicas convencionais justifica a investigação de outras que possam proporcionar a obtenção dessas proteínas, combinando um menor custo com um elevado grau de pureza. Neste sentido, o objetivo deste trabalho foi avaliar e comparar a eficácia dos adsorventes agarose-CM-Asp-Ni(II), agarose-CM-Asp-Co(II), agarose-CM-Asp-Cu(II) e agarose-CM-Asp-Zn(II) na purificação dos fragmentos Fab de IgG humana policlonal, a partir de uma solução de IgG clivada pela enzima papaína. O efeito do sistema tamponante, do íon metálico e do cloreto de sódio foram avaliados. A seletividade das condições cromatográficas foi avaliada por eletroforese SDS-PAGE, Western Blotting e imunodifusão radial. Os resultados indicaram que nas cromatografias conduzidas com o quelato CM-Asp-Co(II) com Hepes e Tris-HCl, na ausência de sal, os fragmentos Fab foram obtidos separados do Fc nas frações de eluição. Nas cromatografias em CM-Asp-Ni(II) com Hepes e fosfato de sódio na presença de NaCl e em CM-Asp-Cu(II) com Tris-HCl e fosfato de sódio na presença de NaCl, a biomolécula alvo foi obtida seletivamente nas frações não retidas (cromatografia negativa). Nas cromatografias em CM-Asp-Zn(II) não houve a recuperação seletiva dos fragmentos Fab. Os resultados das curvas de ruptura com os quelatos CM-Asp-Ni(II), CM-Asp-Co(II) e CM-Asp-Cu(II) revelaram a recuperação de 3,4 mg, 17,1 mg e 8,6 mg de Fab, respectivamente. Os fragmentos Fab foram obtidos com pureza superior a 90%, sendo que para o ligante CM-Asp-Cu(II), segundo o western blot, os fragmentos Fab foram separados da IgG não clivada. Os resultados obtidos nas condições cromatográficas estudadas evidenciam a potencialidade do emprego dos quelatos CM-Asp-Ni(II), CM-Asp-Co(II) e CM-Asp-Cu(II), imobilizados em agarose, para purificação de fragmentos Fab obtidos da clivagem enzimática da IgG humana policlonal / Abstract: Immunoglobulins G (IgG) and their fragments Fab, F(ab)¿2 and Fv are prominently applied as therapeutic and diagnostic tool. Fragments are preferably used rather than the uncleaved IgG, specially when the Fc portion is dispensable or prejudicial. For the mentioned applications, high purity preparations are required. The high costs associated with the conventional downstream processing of these biomolecules is the driving force to investigate purification techniques that can combine lower cost with high purification factor. Therefore, the goal of this work is to evaluate and compare the efficiency of CM-Asp-Ni(II), CM-Asp-Co(II), CM-Asp-Cu(II) and CM-Asp-Zn(II) adsorbents in the purification of Fab fragments from papain-digested human IgG. The effects of buffers, metal-ion and NaCl addition were also studied. The adsorbent/buffer selectivity towards the targeted molecule was evaluated by SDS-PAGE, Western Blotting and radial immunodiffusion assays. Chromatography with CM-Asp-Co(II) as chelated using Hepes and Tris-HCl buffers resulted in Fab recovery in the elution fractions. Whereas the chromatography with CM-Asp-Ni(II) and CM-Asp-Cu(II) both as chelated using Hepes sodium phosphate buffers with NaCl addition resulted in Fab selective recovery in non-retained fractions (negative chromatography). The adsorbent CM-Asp-Zn(II) did not show Fab selective recovery. Breakthrough curves experiments with CM-Asp-Ni(II), CM-Asp-Co(II) e CM-Asp-Cu(II) showed recovery of 3.4 mg, 17.1 mg and 8.6 mg of Fab, respectively. Fab fragments were recovered with purity higher than 90%. When chelated CM-Asp-Cu(II) was use as ligand, Fab fragments were recovery separated from intact IgG as shown in western blot. Results obtained in the chromatographic conditions studied showed the potential use of CM-Asp-Ni(II), CM-Asp-Co(II) and CM-Asp-Cu(II) immobilized in agarose for the purification of Fab fragments from cleaved human polyclonal IgG / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de \"western blot\" para detectar as cadeias de FVIII ou degradação. / Coagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography, followed by gel filtration. The purification process was analyzed by determination of FVIII specific activity and detection of other coagulation factors co eluting in chromatographic fractions. Fragments of FVIII gene were cloned and protein fragments were expressed for animal immunization. Sera with polyclonal antibodies anti-FVIII were used in western blots assays to detect FVIII chains or its degradation.

Mycobacterium tuberculosis, the principal causative agent of tuberculosis (TB) in humans, is considered to be a successful pathogen owing to the elicitation of multidrug resistance, ability to survive inside macrophage phagosomes by taking nutrients from host cell cytoplasm, and the capacity to alternate between proliferating and dormant (nonproliferating) conditions of growth. Thus, whether one looks at tubercle bacillus from the standpoint of regulation of cell division in the host system, or uptake of nutrients from the host cell cytoplasm or elicitation of drug resistance, the requirement for ATP Binding Cassette (ABC) transporter type protein complexes, which might be involved in the transport of drugs, nutrients or proteins, could be of critical importance to the pathogen. Therefore the present study was initiated to characterize ABC transporter type proteins, FtsE and FtsX of M. tuberculosis (MtFtsE and MtFtsX), and their interaction with FtsZ and FtsQ, which are the septation proteins that are recruited respectively before and after the localization of FtsE and FtsX proteins. The study was carried out in 3 parts. 1. Cloning, overexpression and purification of MtFtsE and MtFtsX proteins and elucidation of ATP binding activity of MtFtsE There exists considerable extent of homology between the FtsE and FtsX proteins of M. tuberculosis and E. coli. Therefore, in order to verify whether the structural homology is reflected in functional homology, complementation of growth defect of E. coli ftsE (Ts) by MtFtsE and MtFtsX was carried out. The MtFtsE protein could partially complement growth defect of E. coli ftsE temperature sensitive strain MFT1181, whereas co-expression of MtFtsE and MtFtsX efficiently complemented growth defect, indicating that the MtFtsE and MtFtsX proteins functionally complement E. coli FtsE and FtsX and that the two proteins together might be performing an associated function. Subsequently, in order to biochemically characterize MtFtsE and MtFtsX proteins of M. tuberculosis, MtftsE gene was cloned in pQE30, overexpressed, purified by Ni2+-NTA agarose affinity chromatography under denaturing conditions and refolded. MtFtsX protein, being toxic to E. coli cells, could not be expressed to sufficient amounts. Western blotting with anti-MtFtsE antibody showed that the recombinant 6xHis-MtFtsE protein and the native MtFtsE protein were localized to the membrane of E. coli and M. tuberculosis cells respectively. 6xHis-MtFtsE protein showed ATP binding in vitro, whereas K42R mutation abolished ATP binding. Thus, like in the case of E. coli FtsE, the K42 residue, which is positionally equivalent to K41 in EcFtsE in Walker A motif, was found to be essential for ATP binding. At 1.3 nM concentration of [α32P] ATP,70 molar excess of ATP, ADP, AMP, and GTP competed out respectively 97%, 87%, 73% and 57% of the [α32P] ATP bound to 6xHis-MtFtsE. 2. Biochemical characterization of MtFtsE protein The functional architecture of an ABC transporter consists of two each of nucleotide binding domain (NBD) and transmembrane domain (TMD), which are either part of a single polypeptide chain or individual subunits. The functional NBD is a ‘nucleotide-sandwich dimer’ with ATP flanked by the Walker A and B motifs of one NBD and the signature motif and D-loop of the other. NBD, through ATPase activity, is involved in energizing the transport of substrates namely drugs, proteins, ions, and solutes across the membrane. Since MtFtsE possesses Walker A and Walker B motifs that constitute NBD, and MtFtsX possesses TMD (four transmembrane segments), the two proteins together might constitute an ABC transporter type complex. Therefore, we wanted to know whether MtFtsE could hydrolyze ATP. MtFtsE not only could bind ATP with high affinity but could hydrolyse it also (Km, 1.5 µM; Vmax, 0.87 nmole/mg/min). It could bind and hydrolyse GTP as well, but not CTP, albeit with lower affinity and rate (Km, 25 µM; Vmax, 0.54 nmole/mg/min). The ATPase activity is strongly dependent on Mg2+ or Mn2+, with a pH optimum of 6.5 – 8.0 and temperature range of 27oC - 40oC. Kinetic analysis of ATPase and GTPase activities indicated nucleotide- dependent cooperativity (Hill coefficient for ATP is 1.7 and for GTP, 2.1). Inhibition of ATPase activity, to almost similar extent, in the presence of 10-fold excess of ATPγS, ADP, AMP, GTP, and CTP, but not TTP, indicated that nucleotide binding is through nitrogenous base of the nucleotide. Inhibition of MtFtsE by orthovanadate classified the enzyme as a P-type ATPase. Partially purified MtFtsE in soluble fraction also showed ATPase activity. The ATPase-active form of MtFtsE is a dimer with the sole cysteine (C84) at the dimer interface. Homology modeling of MtFtsE, using MalK (the NBD component of an ABC transporter for maltose) as the template, supported this observation. Stabilization of the dimer through cys-cys disulphide bond increased ATPase activity by 3.7-fold, although C84 does not have any role in ATPase activity. 3. Identification and elucidation of interaction among cell division proteins FtsE, FtsX, FtsQ and FtsZ of Mycobacterium tuberculosis Septum synthesis in E. coli is mediated by a dozen of proteins, among which the bacterial cytoskeletal protein FtsZ is the first molecule to localise to the mid-cell site, where it forms a scaffold for the localization of downstream cell division proteins namely, FtsA /ZipA < FtsE / FtsX < FtsK < FtsQ < FtsL < FtsB < FtsW < FtsI < FtsN and AmiC. If the above order of recruitment of proteins holds true for M. tuberculosis as well, the immediate proteins recruited to the mid-cell site after MtFtsZ in M. tuberculosis would be MtFtsE and MtFtsX, followed with MtFtsK and MtFtsQ. Thus it is possible that MtFtsE and MtFtsX could be interacting with MtFtsZ and MtFtsQ. Therefore attempts were made to delineate the interaction network among MtFtsE, MtFtsX, MtFtsQ and MtFtsZ of M. tuberculosis. Ni2+-NTA agarose pulldown, co-immunoprecipitation and bacterial two-hybrid assays using wild type and deletion mutants of the proteins showed that MtFtsE interacts with MtFtsQ and MtFtsX through its C-terminus. In addition, MtFtsX could interact with MtFtsZ and MtFtsQ. MtFtsX was found to homodimerise and interact with MtFtsQ in vivo. The ATPase-active of MtFtsE in vivo being a dimer, a hypothetical model for the translocation of MtFtsQ into the membrane at mid-cell site was proposed. According to this model, MtFtsQ might be inserted into the membrane at the mid-cell site by (MtFtsX)2 functioning as the membrane channel for the transport, which could be energized by the ATPase subunit (MtFtsE)2 of the (MtFtsE)2(MtFtsX)2 complex. MtFtsX might have a role in tethering the FtsZ-ring with the membrane at the mid-cell site. An altogether different possibility could be that the (FtsE)2(FtsX)2 complex might have a role in the stabilization or constriction of FtsZ-ring during the inward growth of septum.

O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de \"western blot\" para detectar as cadeias de FVIII ou degradação. / Coagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography, followed by gel filtration. The purification process was analyzed by determination of FVIII specific activity and detection of other coagulation factors co eluting in chromatographic fractions. Fragments of FVIII gene were cloned and protein fragments were expressed for animal immunization. Sera with polyclonal antibodies anti-FVIII were used in western blots assays to detect FVIII chains or its degradation.