Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-25Bitstream added on 2014-06-13T20:29:48Z : No. of bitstreams: 1 pesquero_nc_me_araiq.pdf: 1328191 bytes, checksum: b43c0dd0e4c51db5570dd9acd342760e (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / No presente trabalho foi avaliada a equivalência entre as formas nativa e recombinante da lectina Artin M utilizando como ligante a peroxidase de raiz forte (HRP) por meio da técnica de microbalança a cristal de quartzo. Para tal foi preparado um biossensor por meio da imobilização da lectina, tanto nativa como recombinante, na superfície do cristal de quartzo piezelétrico. A imobilização das lectinas foi realizada por meio da construção de uma monocamada auto organizada utilizando dois alcanotióis, ácido 11-mercaptoundecanóico e o 2-mercaptoetanol. Para o acoplamento das proteínas foram utilizados N-etil-N- (dimetilaminopropil) carbodiimida (EDC) e N-hidoxisuccinimida (NHS) que formam com os grupamentos carboxílicos um intermediário reativo. Após a preparação do biossensor foi utilizado um sistema de injeção em fluxo acoplado à microbalança de cristal a quartzo para o estudo da interação lectina-glicoconjugado. Desta forma, as interações da Artin M nativa e recombinante com a glicoproteína peroxidase de raiz forte foram estudadas por meio da determinação das suas constantes de afinidade aparente e de associação cinética, sendo que foram encontrados os valores de constante de afinidade aparente (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP, e os valores de constante cinética (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP. Os valores das constantes de interação obtidos evidenciaram a equivalência entre ambas as formas da lectina Artin M. Neste trabalho também foi determinada a constante de associação cinética da interação entre a lectina Artin M recombinante e linhagens celulares de leucemia mielóide aguda humana (NB4, K562 e U937), no intuito de melhor entender a diferença na citotoxicidade observada da Artin M sobre estas células... / In the present work was evaluated the equivalence between the native and recombinant forms of Artin M using horseradish peroxidase as ligand by means the quartz crystal microbalance technique. In this way, a biosensor was prepared immobilizing the lectin, native and recombinant forms, on piezoelectric quartz crystal surface. Lectins immobilization was realized constructing self assembled monolayers using the alkanethiols 11-mercaptoundecanoic acid and 2-mercaptoethanol. To the binding of proteins was used N-ethyl-N-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which form with carboxylic groups a reactive intermediary. After biosensor preparation was utilized a flow injection system coupled to quartz crystal microbalance to study the lectin-glycoconjugated interaction. Thus the native Artin M and recombinant Artin M interaction with horseradish peroxidase glycoprotein were studied by determining its apparent affinity constant and association kinetic constants. The values obtained to apparent affinity constant were (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions, and the values obtained to association kinetic constant were (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions. These constant values evidence the equivalence between native and recombinant forms of Artin M lectin. During this work were also determined the association kinetic constant of the interaction between recombinant Artin M and leukemic lineages from human acute myeloid leukemia (NB4, K562 and U937), with the purpose of a better understanding in the different cytotoxic effect of Artin M on these cells. In this way the values of association kinetic constant obtained was (0,3 ± 0,1) x 10-7 , (0,9 ± 0,1) x 10-7 e (2,7 ± 0,3) x 10-7 mL cel -1 to the interactions between Artin M and NB4, K562 and U937, respectively

Orientador: Paulo Roberto Bueno / Banca: Marcelo Mulato / Banca: Maria Cristina Roque Antunes Barreira / Resumo: No presente trabalho foi avaliada a equivalência entre as formas nativa e recombinante da lectina Artin M utilizando como ligante a peroxidase de raiz forte (HRP) por meio da técnica de microbalança a cristal de quartzo. Para tal foi preparado um biossensor por meio da imobilização da lectina, tanto nativa como recombinante, na superfície do cristal de quartzo piezelétrico. A imobilização das lectinas foi realizada por meio da construção de uma monocamada auto organizada utilizando dois alcanotióis, ácido 11-mercaptoundecanóico e o 2-mercaptoetanol. Para o acoplamento das proteínas foram utilizados N-etil-N- (dimetilaminopropil) carbodiimida (EDC) e N-hidoxisuccinimida (NHS) que formam com os grupamentos carboxílicos um intermediário reativo. Após a preparação do biossensor foi utilizado um sistema de injeção em fluxo acoplado à microbalança de cristal a quartzo para o estudo da interação lectina-glicoconjugado. Desta forma, as interações da Artin M nativa e recombinante com a glicoproteína peroxidase de raiz forte foram estudadas por meio da determinação das suas constantes de afinidade aparente e de associação cinética, sendo que foram encontrados os valores de constante de afinidade aparente (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP, e os valores de constante cinética (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP. Os valores das constantes de interação obtidos evidenciaram a equivalência entre ambas as formas da lectina Artin M. Neste trabalho também foi determinada a constante de associação cinética da interação entre a lectina Artin M recombinante e linhagens celulares de leucemia mielóide aguda humana (NB4, K562 e U937), no intuito de melhor entender a diferença na citotoxicidade observada da Artin M sobre estas células ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the present work was evaluated the equivalence between the native and recombinant forms of Artin M using horseradish peroxidase as ligand by means the quartz crystal microbalance technique. In this way, a biosensor was prepared immobilizing the lectin, native and recombinant forms, on piezoelectric quartz crystal surface. Lectins immobilization was realized constructing self assembled monolayers using the alkanethiols 11-mercaptoundecanoic acid and 2-mercaptoethanol. To the binding of proteins was used N-ethyl-N-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which form with carboxylic groups a reactive intermediary. After biosensor preparation was utilized a flow injection system coupled to quartz crystal microbalance to study the lectin-glycoconjugated interaction. Thus the native Artin M and recombinant Artin M interaction with horseradish peroxidase glycoprotein were studied by determining its apparent affinity constant and association kinetic constants. The values obtained to apparent affinity constant were (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions, and the values obtained to association kinetic constant were (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions. These constant values evidence the equivalence between native and recombinant forms of Artin M lectin. During this work were also determined the association kinetic constant of the interaction between recombinant Artin M and leukemic lineages from human acute myeloid leukemia (NB4, K562 and U937), with the purpose of a better understanding in the different cytotoxic effect of Artin M on these cells. In this way the values of association kinetic constant obtained was (0,3 ± 0,1) x 10-7 , (0,9 ± 0,1) x 10-7 e (2,7 ± 0,3) x 10-7 mL cel -1 to the interactions between Artin M and NB4, K562 and U937, respectively / Mestre

Ticks are blood feeding parasites that secrete a number of immunomodulatory factors to evade host immune response. The aim of this study was to prepare a tick salivary protein with anti-chemokine activity and to observe the influence of salivary gland extrakt on neutrophile´s chemotaxis.

The spun-out silk fiber consists of two fibroin filaments that are cemented together by sericin coating. The serine-rich sericins, which make 20-30% of the cocoon silk proteins in Bombyx mori, are dissolved in hot water during silk fiber reeling from the cocoon. The sericin extract is usually discarded. Only small amounts are currently used in cosmetics and lately also as replacement of bovine serum products in the cell culture media. The use in culture media is hindered by poor standardization of the extracts. To overcome this problem, we attempted preparation of several recombinant proteins based on partial sequences of one out of the three sericin genes of Bombyx mori, sericin 3. Translation product of the Ser3 gene is extremely serine-rich with a relatively high representation of the aspartate, asparagin, glutamate, and glutamine. Using specific primers we have prepared a cDNAs of 267, 279, 525, 672, and 528 bp, respectively, derived from the Ser3 gene. The cDNAs were cloned and expressed as fusion proteins with hexahistidine in Escherichia coli. The proteins were purified by affinity chromatography and analyzed by acrylamide electrophoresis. Ser3 gene contain repetitive motifs rich in serine and including some charged amino acids.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / As lectinas constituem ferramentas biotecnolÃgicas de fundamental importÃncia em estudos histoquÃmicos e citoquÃmicos para a detecÃÃo de glicoconjugados nos tecidos. Entre estas aplicaÃÃes, destacam-se a identificaÃÃo de receptores de membrana e a detecÃÃo de estruturas caracterÃsticas de neoplasias. A Frutalina, uma lectina -D-galactose-ligante encontrada em sementes de Artocarpus incisa, jà foi utilizada na detecÃÃo histoquÃmica de lesÃes malignas de mama e tireÃide. No presente trabalho foi realizada a clonagem e expressÃo do gene da frutalina em cÃlulas de Escherichia coli Origami (DE3) e foi determinada a estrutura tridimensional dessa proteÃna atravÃs da modelagem por homologia. A clonagem do gene da frutalina foi realizada a partir do produto amplificado da RT-PCR de sementes em diferentes estÃgios de maturaÃÃo dos frutos de A. incisa. Foram obtidos 12 clones, dos quais 5 foram seqÃenciados. A lectina recombinante foi expressa como cadeia Ãnica, formada pela cadeia beta com 20 resÃduos, ligada a um peptÃdeo de ligaÃÃo com quatro resÃduos, e a cadeia alfa com 133 resÃduos. A expressÃo da lectina foi indicada pelo aparecimento de uma banda protÃica com massa molecular aparente de 19,2 kDa, superior a da lectina nativa (15 kDa), apÃs a induÃÃo com IPTG 1mM. A lectina recombinante foi mantida exclusivamente em corpos de inclusÃo e foi reconhecida imunologicamente por anticorpos policlonais anti-Frutalina. Os modelos gerados para os clones obtidos indicam que as mutaÃÃes ocorridas nÃo alteram os sÃtios de ligaÃÃo a carboidratos das molÃculas. As mutaÃÃes nos clones obtidos sugerem a ocorrÃncia de isoformas dessa proteÃna. / The lectins constitute biotechnological tools of fundamental importance in hystochemical and cytochemical studies for the detection of tissue glycoconjugates. Among their applications are the identification of membrane receptors and the detection of neoplastic characteristic structures. Frutalin, an -D-galactose-binding lectin from Artocarpus incise seeds, has already been used in the hystochemical detection of thyreoid and breast neoplasia. In this work was made a study of the cloning and expression of the frutalin gene in Escherichia coli Origami (DE3) cells and was determined the three-dimensional structure of this protein through homology modeling. The frutalin gene cloning was accomplished from the product on RT-PCR with seeds in distinct stages of development of the Artocarpus incisa fruits. Twelve clones were obtained, from which five were sequenced. The frutalin gene was expressed in E. coli using the pET15b expression vector. A recombinant lectin (rFrutalin) was expressed by growing the bacteria in the presence of isopropyl -D-thiogalactopyranoside 1mM. All the recombinant lectin was found in an insoluble aggregated form as inclusion bodies. The recombinant lectin had a higher molecular mass (19,2 kDa) than the native lectin (15 kDa) as estimated by SDS-PAGE and Western blot analyses, showing that the recombinant single chain Frutalin is not processed in E. coli cells. The models generated for the clones obtained indicate that the mutations do not change the molecules active sites. Mutations in the clones obtained suggest the occurrence of isoforms of this protein.

Made available in DSpace on 2015-04-11T13:55:02Z (GMT). No. of bitstreams: 1 Luciana Pereira de Sousa.pdf: 2276123 bytes, checksum: a079176d073fb54e951e38fb4291c529 (MD5) Previous issue date: 2012-11-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Malaria is one of the most serious public health problems in the world, accounting for high morbidity and mortality. The diagnosis remains the most widely used microscopy, however this approach requires adequate infrastructure and highly skilled professionals for the exams, making it unfeasible in areas of difficult access. Tests for the rapid and simple diagnosis of malaria are commercially available, but none of national origin, complicating the deployment by the Unified Health System (SUS). Faced with this problem, this study has as main objective the production of Plasmodium vivax Lactate Dehydrogenase (pLDH), aiming at the development of polyclonal antibodies anti-LDH able to detect the native antigen in blood samples from patients infected with the intention of offering future perspectives for the development of a rapid diagnostic test for malaria. For this, pvLDH protein was produced by recombinant DNA technology in host cells chemically competent. Therefore, it was purified to inoculation into rabbits and mice Balb /c. The response of the inoculated animals as well as the performance of polyclonal antibodies anti-LDH in the recognition of native antigen were assessed by ELISA immunoassays and sandwich system, respectively. The animals showed good antibody titers anti-pLDH after the third inoculation of the recombinant protein pLDH, and the rabbit antibody response than the response of mice with absorbance values at dilution 1/100, 2,600 and 2,100, respectively. Polyclonal antibodies anti-pLDH were able to recognize the native protein pLDH 131 samples to 154 samples positive malaria and incapable of recognizing human isoforms of LDH when evaluated against malaria negative samples, since the 23 negative samples evaluated, all also remained negative during the tests. The proposal idealized in this study proved extremely viable and promising. The results of this study meet expectations and provided future prospects as regards the use of recombinant pvLDH for the development of polyclonal and monoclonal antibodies functional in murine model for use in rapid diagnostic test (RDT) for malaria in attempt to offer alternatives to diagnosis in Brazil, or for use in immunoassay targeting the monitoring of the disease course. / A malária é um dos mais sérios problemas de saúde pública do mundo, sendo responsável por elevada morbidade e mortalidade. O diagnóstico mais utilizado continua sendo a microscopia, contudo esta metodologia exige infraestrutura adequada e profissionais altamente qualificados para a realização dos exames, tornando-se inviável em áreas de difícil acesso. Testes para o diagnóstico rápido e simples de malária estão disponíveis no mercado, porém nenhum de origem nacional, dificultando a implantação pelo Sistema Único de Saúde (SUS). Diante desta problemática, o presente estudo tem como principal objetivo a produção de Lactato Desidrogenase de Plasmodium vivax (pvLDH), visando o desenvolvimento de anticorpo os policlonais anti-LDH capazes de detectar o antígeno nativo em amostras sanguíneas de pacientes infectados com a pretensão de oferecer perspectivas para a elaboração de um teste diagnóstico rápido para a Malária. Para isto, a proteína pvLDH foi produzida pela tecnologia do DNA recombinante em células hospedeiras quimicamente competentes. Logo, a proteína foi purificada para a imunização de coelho e camundongos Balb/c. A resposta dos animais as inoculações assim como o desempenho dos anticorpos policlonais anti-LDH no reconhecimento do antígeno nativo foram avaliados por ensaios imunoenzimáticos ELISA indireto e em sistema sanduíche, respectivamente. Os animais demonstraram boa resposta de anticorpos anti-pvLDH após a terceira imunização de proteína recombinante, sendo a resposta de anticorpos do coelho superior a resposta dos camundongos, com valores de absorbância de até 2.600 e 2.100, respectivamente. Os anticorpos policlonais anti-pvLDH foram capazes de reconhecer a proteína LDH nativa em 131 amostras de 154 amostras positivas para malária e incapazes de reconhecer isoformas de LDH humana quando avaliados em relação a amostras negativas para malária, uma vez que das 24 amostras negativas avaliadas, todas se mantiveram também negativas nos ensaios. A proposta idealizada neste trabalho se mostrou extremamente viável e promissora. Os resultados obtidos neste estudo corresponderam às expectativas e forneceram perspectivas futuras no que diz respeito à utilização da pvLDH recombinante para a produção de anticorpos policlonais e monoclonais funcionais, em modelo murino, para a utilização em teste diagnóstico rápido (TDR) para a malária, na tentativa de oferecer alternativas ao diagnóstico no Brasil, ou para a utilização em imunoensaio visando o monitoramento do curso da doença.

O P. vivax é a segunda espécie mais prevalente causadora de malária no mundo. Medidas de controle ineficientes exigem o desenvolvimento de novas estratégias de prevenção, como vacinas, novas drogas e novos inseticidas. O objetivo geral do trabalho foi gerar uma formulação vacinal universal com proteínas e adenovírus recombinantes capazes de induzir anticorpos contra as diferentes formas alélicas da proteína circumsporozoíta (CSP) do P. vivax. As proteínas foram produzidas em E. coli e purificadas por cromatografia de afinidade e troca iônica. A obtenção destas proteínas nos permitiu testar qual seria a melhor formulação vacinal para a indução de anticorpos contra as três formas alélicas da proteína CSP de P. vivax (PvCSP). Anticorpos específicos reconheceram esporozoítas do P. vivax por imunofluorescência. Por fim testamos o uso de dois adenovírus recombinantes, um símio e um humano, deficientes em replicação, expressando as três regiões imunodominantes da proteína PvCSP em fusão. Estes foram capazes de induzir resposta imune específica contra as proteínas PvCSP sendo testados em esquema de prime-boost heterólogo, onde camundongos foram primados com os adenovírus e nas doses-reforço receberam a mistura com as três proteínas recombinantes. / The Plasmodium vivax is the second most prevalent species of malaria in the world. Inefficient measures of control used today demand the development of new strategies for prevention, as vaccines, new drugs and new insecticides. The central objective of this thesis was to generate a universal vaccine formulation with proteins and recombinant adenoviral vectors representing the different allelic forms of the circumsporozoite protein (CSP) of the P. vivax. The recombinant proteins were expressed in E. coli and purified. These proteins allowed us to test which would be the best vaccine formulation for the induction of antibodies against the three allelic forms of CSP. The specific antibodies also recognized P. vivax sporozoites by immunofluorescence. Finally we test the use of two recombinant adenoviral vectors, a simian and a human, both replication deficient, expressing a protein containing the repeat regions of the CSP in fusion. These adenoviral vectors induced specific immune response against CSP and were successfully used in an immunization regimen of heterologous prime and boost where in the first dose the mice received recombinant adenoviral vector and in the subsequent doses, the mixture with three recombinant proteins.

O Fator VII recombinante (FVIIr) tem sido a principal escolha terapêutica dos pacientes hemofílicos que desenvolvem inibidores contra os fatores VIII e IX utilizados como tratamento. Atualmente, o produto utilizado é produzido em células de camundongo (BHK-21), o qual oferece desvantagens considerando a complexidade das modificações pós-traducionais desta proteína e a inserção de glicosilações de origem murina altamente imunogênicas aos seres humanos. Dessa maneira a produção de proteínas para uso terapêutico em linhagens celulares humanas surge como uma alternativa promissora. Dentro desse contexto, o objetivo principal deste trabalho foi clonar e expressar o FVII de coagulação sanguínea em 3 linhagens celulares humanas (HepG2, Sk-Hep, HKB-11), compará-las com a linhagem murina BKH-21, e selecionar a melhor produtora da proteína recombinante. As células foram modificadas com o vetor lentiviral p1054-CIGWS, contendo os genes do FVII e do marcador GFP. Após a modificação das células foi observada uma eficiência de transdução de 80% nas células BHK-21-FVIIr, 73% nas células HepG2-FVIIr, 32% nas células HKB-11-FVIIr e 95% Sk-Hep-FVIIr. Análises da expressão gênica por PCR em Tempo Real mostraram que as três linhagens humanas modificadas apresentaram expressão do RNAm relativo ao FVIIr, sendo que a linhagem celular HepG2 foi a que teve maior expressão de FVIIr, seguida da Sk-Hep-1 e HKB-11. Quando submetidas ao tratamento com vitamina K por um período de 10 dias em cultura, a expressão do gene FVIIr foi semelhante para as três linhagens (HepG2: 164563 URE, HKB-11: 119122 URE e Sk-Hep: 124919 URE). O FVII é uma proteína que para sua ativação, possui como principal modificação pós traducional a -carboxilação vitamina K dependente, que ocorre por meio do ciclo da vitamina K com a participação de 3 enzimas, -carboxilase, VKORC1 e calumenina (inibidor). A expressão gênica dessas enzimas foi avaliada antes e após o tratamento com a vitamina K. Foi possível observar que houve um aumento nos níveis de RNAm nas células humanas tratadas com vitamina K, sugerindo que esta é capaz de ativar as enzimas do ciclo da -carboxilação. A cinética de crescimento celular em garrafas estáticas mostrou que a as células murinas BHK-21 modificadas possuem uma velocidade específica de crescimento 25% mais elevada que das células humanas. Contudo a cinética de produção das linhagens recombinantes mostrou que as células humanas produzem cerca de 3 vezes mais FVIIr do as células BHK-21. Devida a baixa produção de FVIIr na linhagem celular murina, e ao fato de que a linhagem humana HepG2 apresenta um perfil de crescimento extremamente lento, as linhagens recombinantes Sk-Hep-1-FVIIr e HKB-11-FVIIr foram selecionadas para ensaios de cultivo em suspensão utilizando microcarregadores em frascos spinners. Ao longo de 10 dias de cultivo as células HKB-11-FVIIr mostraram uma produção acumulada de 152 g de FVIIr, o que corresponde a 304 UI. As células Sk-Hep-1-FVIIr produziram cerca de 202,6 g de FVIIr, o que corresponde a 405,2 UI. Em suma, nossos dados comprovam que as linhagens celulares humanas são eficazes para a produção de fator VII recombinante, uma vez que, utilizando nosso modelo de produção, estas mostraram-se melhores do que a de células murinas (BHK-21) utilizadas pela indústria. Assim, estas linhagens celulares humanas podem ser usadas como uma nova plataforma para a produção de FVII, bem como para outras proteínas recombinantes, de maneira mais segura e com menor risco de desenvolvimento de anticorpos inibidores / Recombinant factor VII (FVIIr) has been the main therapeutic choice for hemophilic patients who develop inhibitors antibidies to conventional treatments (FVIII and FIX). Currently, the comercial product is produced in murine cells (BHK-21) which gives disadvantages considering the complexity of post-translational modifications of these proteins. The insertion of murine residues can be highly immunogenic in humans. Thus the production of proteins for therapeutic use in human cell lines appears as a promising alternative. In this context, the aim of this study was to clone and express the blood coagulation FVII in 3 human cell lines (HepG2, Sk-Hep-1, HKB-11) and select the best cell line for production of recombinant protein. The cells were modified with the lentiviral vector p1054-CIGWS containing the FVII gene and GFP gene marker. After cells modification we observed efficiency of transduction, in which 80% of BHK-21-FVIIr cells showed GFP expression, 73% of HepG2-FVIIr cells, 32% of HKB-11-FVIIr cells and 95% SK- Hep-FVIIr. Gene expression analysis by real-time PCR showed that the three modified human cell lines exhibited RNAm expression relative to FVIIr. When cells were treated with 5 ug/mL vitamin K in culture, the gene expression of FVIIr was similar in the three cell lines (HepG2: 164 563 URE, HKB-11: 119122 and SK-Hep URE: 124919 URE). For FVII activation, the main post translational modification is -carboxylation vitamin-K-dependent which envolves three enzymes, -carboxylase, VKORC1 and calumenina (inhibitor) . Gene expression of these enzymes was evaluated before and after treatment with vitamin K. It was observed that there was an increase in mRNA levels in human cells treated with vitamin K, suggesting that the treatment is capable of activating the enzymes of the vitamin K cycle. Cell growth kinetics showed that modified murine cells BHK-21 have a higher specific growth rate, around 25% more than human cells. However the kinetics of production of recombinant cell lines showed that human cells expressing rFVII 3-fold more rFVII than BHK-21 cells. Due the low rFVII production of murine cells, and the extremely slow growth profile of human cell line HepG2, the recombinant cell lines Sk-Hep-1-rFVII and HKB-11-rFVII have been selected for cultivation tests in suspension using microcarriers in spinners flasks. Over 10 days of cultivation the HKB-11 cells showed a cumulative production of rFVII 152 ug, corresponding to 304 IU and SK-Hep-1 cells showed a rFVII production of 202.6 ug, corresponding to 405.2 IU. In summary, our data demonstrate that human cell lines are effective for producing recombinant factor VII. Using our production model, human cells were better than murine cells (BHK-21) used by the industry. Thus, these human cell lines can be used as a new platform for the FVII production, as well as for other recombinant proteins, with less risk of developing inhibitor antibodies

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

Abstract Collagen prolyl 4-hydroxylase (C-P4H) plays a central role in the biosynthesis of collagens by hydroxylating proline residues. The enzyme has been a subject of intense interest as a target enzyme for drug development. The recombinant expression of human C-P4H in prokaryotes has not yet been described. This work reports on the development of an expression system for human C-P4H in E. coli. The vertebrate C-P4H enzymes are α2β2 tetramers, consisting of two β subunits which are identical to protein disulphide isomerase (PDI), aside from the two α subunits which have the catalytic activity. The function of PDI is to keep the α subunit in a soluble and active state. Therefore, the expression system should assure the expression of the β subunit in the cell before the α subunit by using two different promoters. An active C-P4H tetramer was obtained in the periplasm of E. coli. However, further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human C-P4H tetramer. The exchange of four rare E. coli codons of the pdi gene and the optimized distance between ribosome binding site and translation initiation, resulted in 50-fold P4H-activity and 25 mg/l purified enzyme. Comparison of the expression level of mRNA from the α and β subunits by Sandwich hybridization identified single induction with anhydrotetracycline in fed-batch fermentations as a limiting parameter. This caused an insufficient expression level of mRNA and thereby a low yield of C-P4H. A maximum yield was obtained by repeated addition of anhydrotetracycline that led to higher mRNA levels and increased productivity. A newly developed stochastic simulation model of translational ribosome traffic in bacteria assesses the effect of codon usage to ribosome traffic and to the overall translation rate and mRNA stability. Using human PDI, it was shown that substitution of four 5' codons of the human PDI sequence that are rare in E. coli sequences, by synonymous codons preferred in E. coli led to a 2-fold increase of total PDI amount and even to a 10-fold increase of soluble PDI amount.