Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_silvana_marchioro.pdf: 699240 bytes, checksum: 32b6c311c57b68941cfdd0ee832643a1 (MD5) Previous issue date: 2008-11-27 / Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), one the most common respiratory diseases of pigs worldwide. The disease is characterized by retarded growth rate in animals of mid-finishing to slaughter age, causing significant economic losses to swine production. Vaccination is the most cost-effective strategy for the control of this disease. However, the vaccines against the disease available on the market have a high production cost and only offer partial protection, not preventing the establishment from infection and the presence of pigs carriers. This issue highlights the need for the use of new strategies aiming at circumventing this problem. The recombinant DNA technology is an important tool for the development of vaccines. The publication of the sequence of three genomes of M. hyopneumoniae, two pathogenic strains (232 and 7448) and a non-pathogenic (J) is helping the researches, and accelerating the process of identification of new antigenic proteins. From the sequence data and proteomic analysis, it was possible to identify coding sequences (CDS) of transmembrane proteins. These antigens were cloned and expressed using E. coli as an expression system. A total of seven recombinant proteins were purified and evaluated regarding immunogenicity in mice and antigenicity with sera from animals with EP. All the antigens evaluated were able to induce a significant immune response in mice. The evaluation of antigenicity properties revealed the proteins reacted with sera from pigs infected with M. hyopneumoniae. The recombinant proteins evaluated in this study may be of value to use in the development of vaccines and tests for immunodiagnosis of swine enzootic pneumonia. / Mycoplasma hyopneumoniae é o agente etiológico da pneumonia enzoótica suína (PES), uma das doenças respiratórias de suíno mais comum em todo o mundo. Ela é caracterizada por retardo no desenvolvimento em animais na fase de crescimento e terminação, causando perdas econômicas consideráveis na indústria suinícola. A vacinação parece ser a forma mais efetiva de controlar a PES, entretanto, as vacinas contra a doença disponíveis no mercado apresentam um elevado custo de produção e conferem uma proteção parcial, não impedindo o estabelecimento da infecção e nem a presença de suínos portadores. Desta forma, surge à necessidade da utilização de novas estratégias visando contornar este problema. A tecnologia do DNA recombinante constitui uma ferramenta importante para o desenvolvimento de vacinas. As publicações das seqüências de três genomas de M. hyopneumoniae, duas cepas patogênicas (232 e 7448) e uma não patogênica (J) estão contribuindo com pesquisas e, acelerando o processo de identificação de novas proteínas imunogênicas. A partir dos dados gerados e da análise proteômica complementar, foi possível a identificação de seqüências codificadoras (CDS) de proteínas transmembrana. Estas seqüências foram clonadas e expressas em E. coli. Um total de sete proteínas recombinantes foram purificadas e avaliadas quanto à imunogenicidade em camundongos e à antigenicidade frente a soros de suínos com PES. Todas as proteínas avaliadas foram capazes de induzir uma resposta imune significativa em camundongos. O estudo das propriedades antigênicas revelou que estas são reconhecidas pelo soro de suínos infectados com M. hyopneumoniae. As proteínas recombinantes avaliadas neste estudo poderão ser de valia para utilização como vacinas de subunidade e em testes de imunodiagnóstico da PES.

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_leandro_nizoli.pdf: 538110 bytes, checksum: 770c97bed302c0714288a9278ce94694 (MD5) Previous issue date: 2009-03-18 / Equine theileriosis is considered to be one of the most important parasitic diseases that affect horses, and has great economic impact on the equine industry. The disease is caused by the etiologic agent Theileria equi, which is classified as a hematozoan. The losses associated with equine theileriosis are related to clinical manifestation as well as restriction to international travel to positive horses. Chronic infected equines suffer the risk of the disease relapse which leads to losses in reproduction performance and are potentially disseminators of the disease. In the last years, studies on the immunologic diagnosis and vaccination against T. equi have focused to obtain distinct antigenic proteins. On the outer membrane of this protozoan, major surface proteins has been characterized and named as EMAs (equi merozoite antigen). Of these, EMA-1 has been used as antigen for diagnosis due to its conservation in diverse isolates. Its role as a potential immunogen has been well documented due its ability to stimulate a humoral response with production of specific antibodies in infected animals. Through this antibodies one can used as tool for immune diagnostic of this disease. EMA-1 is also a strong candidate to be use as a vaccine in the control of equine theileriosis. In this study we used the Pichia pastoris yeast as expression system for the production of the EMA-1 protein of T. equi and evaluated its antigenicity and immunogenicity. When tested for antigenicity, the recombinant protein was recognized by antibodies form chronic T. equi infected horses, suggesting that epitopes of the native were conserved in the recombinant protein. Also we were able to observe that this protein was immunogenic in mice. The data obtained in this study demonstrated that the yeast P. pastoris is an expression system of heterologous protein suitable for the production of EMA-1 from T. equi. / A Theileriose eqüina é considerada uma das principais doenças parasitárias que acometem os eqüinos, acarretando grande impacto econômico na equinocultura. A doença é causada pelo hematozoário Theileria equi. As perdas econômicas associadas à theileriose eqüina estão relacionadas tanto aos fatores clínicos, quanto à restrição ao trânsito internacional de animais soropositivos, já que animais portadores crônicos são passíveis de reagudizações, gerando perda de performance física e reprodutiva, e são potencialmente disseminadores da enfermidade. Nos últimos anos, os estudos sobre o diagnóstico imunológico e vacinação contra T. equi concentram-se na obtenção de frações antigênicas. Na membrana externa deste protozoário foram caracterizadas proteínas principais de superfície denominadas de EMAs (equi merozoite antigen). Dentre estas, a EMA-1 destaca-se como antígeno para diagnóstico em função de sua conservação entre diversos isolados. Seu papel também tem sido caracterizado como imunógeno por estimular forte resposta humoral com produção de anticorpos em animais infectados, podendo ser usado como ferramenta para imunodiagnóstico dessa doença. EMA-1 é também um potencial candidato como antígeno vacinal no controle da theileriose equina. Neste estudo utilizou-se o sistema eucariótico de expressão baseado na levedura metilotrófica Pichia pastoris, para a produção da proteína EMA-1 de T. equi e a avaliação quanto a sua antigenicidade e imunogenicidade. Quanto a sua antigenicidade, a proteína recombinante foi reconhecida por anticorpos de animais portadores crônicos de T. equi, sugerindo que epítopos nativos foram conservados na proteína recombinante. Também foi observado que a proteína recombinante foi capaz de gerar resposta imune em camundongos vacinados com esta proteína. Os dados obtidos neste estudo demonstram que a levedura P. pastoris é um sistema de expressão heterólogo adequado para a produção da proteína EMA-1 de T. equi, podendo ser utilizada como imunobiológico no desenvolvimento de testes diagnósticos e vacina recombinante.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bovine herpesvirus type 1 (BoHV-1) is distributed worldwide and produces high economic losses to the in livestock industry. BoHV-1 infection causes respiratory, reproductive and may also be associated with neurological signs. There are several tests that can diagnose the infection, however, serological techniques currently used are not able to differentiate antibodies produced by vaccination from those produced in response to natural infection. What is sought is a mean to differentiate vaccinated animals of those infected by the field strain. Vaccines with deletion in the glycoprotein E (gE) gene have been developed for this purpose. However, this also requires the development of tests capable to differentiate the serological response between infected and vaccinated animals. To this end, a 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of the BoHV-1 gE gene - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector pET16b. A soluble protein of approximately 25 kDa was purified from lysates of transformed E. coli. The recombinant protein was detected in Western blot (WB) by anti-6-his tag and anti BoHV-1 gE monoclonal antibodies. Antibodies present in the sera of cattle infected with BoHV-1 and BoHV-5 reacted specifically with the 25 kDa recombinant protein in WB. Moreover, mice immunized with the purified protein developed antibodies that recognized the viral gE in lysates of cell monolayers infected with BoHV-1 and BoHV-5. An indirect ELISA for gE antibodies, based on the expressed protein, was able to differentiate serologically calves vaccinated with a gE-deleted BoHV- 5 strain from calves experimentally infected with BoHV-1 or BoHV-5. These data demonstrate that the antigen retained its immunological properties and, thus, can be used in serological tests for bovine herpesvirus infections. It has a potential use in a indirect ELISA to differentiate naturally infected animals from those vaccinated whit the recombinant, gE-negative strains. / O herpesvírus bovino tipo 1 (BoHV-1) é um vírus de distribuição mundial e produz grandes prejuízos econômicos em rebanhos de corte e de leite. A infecção pelo BoHV-1 produz manifestações respiratórias, reprodutivas e também pode cursar com sinais nervosos. Existem diversos testes laboratoriais capazes de diagnosticar a infecção. Contudo, as técnicas sorológicas empregadas atualmente não são capazes de diferenciar anticorpos produzidos pela vacinação daqueles produzidos em resposta à infecção natural. Assim, vacinas diferenciais com deleção da glicoproteína E (gE) têm sido desenvolvidas com essa finalidade. No entanto, necessita-se também de testes capazes de diferenciar a produção de anticorpos vacinais dos induzidos pelo vírus vacinal. Com essa finalidade, essa dissertação relata a expressão e caracterização de um fragmento da glicoproteína E do BoHV-1 e seu uso no desenvolvimento e padronização de um ELISA indireto para detecção de anticorpos anti-gE. Um fragmento de 651 nucleotídeos correspondente ao terço amino-terminal (217 aminoácidos) do gene da gE do BoHV-1, que possui uma alta identidade com o homólogo herpesvírus bovino tipo 5 (BoHV-5), foi clonado com proteína de fusão 6xHis-tag em Escherichia coli utilizando vetor de expressão pET16b. Uma proteína solúvel de aproximadamente 25kDa foi purificada a partir de lisados de E. coli transformadas. A proteína recombinante foi detectada por Western blot (WB) por anticorpos monoclonais anti-histidina e anti-gE do BoHV-1. Anticorpos presentes no soro de animais infectados com BoHV-1 e BoHV-5 reagiram especificamente com a proteína recombinante no WB. Além disso, camundongos imunizados com a proteína purificada desenvolveram anticorpos que reconheceram a gE viral proveniente de lisados de monocamadas celulares infectadas com BoHV-1 e BoHV-5. Um ELISA indireto para detecção de anticorpos anti-gE, baseado na proteína expressa, foi capaz de diferenciar sorologicamente animais vacinados com a cepa gE deletada do BoHV-5 dos animais experimentalmente infectados com BoHV-1 ou BoHV-5. Esses resultados demonstram que o antígeno obtido conservou suas características imunológicas e pode ser utilizado na detecção sorológica das infecções por herpesvírus bovinos. Possui potencial para uso em grande escala como antígeno em testes de ELISA para diferenciar animais naturalmente infectados de animais vacinados com a cepas defectivas na gE

Abstract Small-scale cultivation methods are a necessity for the development of new biotechnological processes. The most common method for submerged microbial cultivation is a shake flask used with a batch operation protocol. Well plate cultivation formats have also increased their importance, due to the need to utilize high-throughput cultivations for efficient product development. However, batch cultivation is often not the optimal method for obtaining high cell densities and good product quality, due to unlimited microbial growth. The aim of this dissertation was to improve small-scale microbial cultivations for microbial growth and product formation. Hydrolytic enzymes were utilized to relieve nutrient limitation by hydrolysis of proteins in lactic acid bacteria cultures to improve lactic acid production from dairy side products. Hydrolytic enzymes were also utilized in the enzymatic release of glucose from starch to create a fed-batch-like cultivation system applicable on small scale. The wireless sensor system developed was applied in shake flask cultivations to monitor oxygen and pH levels. Enzymatic polymer processing was applicable for small-scale cultivations. Lactic acid production by Lactobacillus salivarius ssp. salicinius was enhanced four-fold when the proteins were hydrolyzed either by proteases or by proteolytic microbes. The fed-batch-mimicking controlled glucose feeding and growth control were obtained by means of the simultaneous enzymatic hydrolysis of starch-polymer during cultivation. Controlled growth, higher cell densities, decreased side product formation and increased amount of soluble protein product were obtained in Escherichia coli cultivations. When this method was applied to the cultivation and recombinant protein production of the methylotrophic yeast Pichia pastoris, higher cell densities and higher amounts of active protein were obtained. The glucose concentration remained low enough to avoid the substrate repression of the alcohol oxidase promoter. The fed-batch method is suitable for high-throughput cultivations since the method can be utilized in well plate formats without external feeding devices. The method can be utilized in the development of new biotechnological products, especially when the production system is sensitive to growth conditions, and growth control is preferred. / Tiivistelmä Pienen mittakaavan mikrobikasvatusmenetelmiä tarvitaan kehitettäessä uusia bioteknologisia prosesseja. Tavallisin menetelmä mikrobien liuoksessa tapahtuvaan kasvatukseen on panostyyppisesti tehtävä sekoituspullokasvatus. Kuoppalevykasvatukset ovat myös tulleet entistä tärkeämmiksi, koska tuotekehityksen tehostamiseksi on tarvetta käyttää high-throughput-menetelmiä. Tavoiteltaessa korkeita mikrobisolutiheyksiä ja tuotteen hyvää laatua, panostyyppinen kasvatus ei ole usein paras vaihtoehto, johtuen mikrobien rajoittamattomasta kasvusta. Tämän työn tarkoituksena oli parantaa mikrobien kasvua ja tuotteen muodostusta pienen mittakaavan kasvatuksissa. Meijeriteollisuuden sivutuotteiden proteiineja pilkottiin entsyymien avulla, jotta maitohappobakteerit pystyivät hyödyntämään proteiinit tehokkaammin ja tuottamaan enemmän maitohappoa. Hydrolyyttisiä entsyymejä hyödynnettiin myös glukoosin vapauttamiseen tärkkelyksestä, jolloin saatiin luotua pieneen mittakaavaan sopiva panossyöttötyyppinen kasvatusmenetelmä. Työn aikana kehitettyä langatonta mittausjärjestelmää hyödynnettiin sekoituspullokasvatuksissa happipitoisuuden ja pH:n seurantaan. Entsymaattinen polymeerien käsittely oli soveltuva menetelmä pienen mittakaavan kasvatuksiin. Maitohapon tuotto Lactobacillus salivarius ssp. salicinius -mikrobilla nelinkertaistui, kun ravinneproteiinit pilkottiin joko proteaasien tai proteolyyttisten mikrobien avulla. Panossyöttömenetelmää muistuttava hallittu glukoosin syöttö ja mikrobin kasvun hallinta saavutettiin pilkkomalla tärkkelystä glukoosiksi kasvatuksen aikana. Escherichia coli kasvatuksissa saavutettiin hallittu solumäärän kasvu, korkeammat solutiheydet, vähentynyt sivutuotteiden muodostus ja suurempi liukoisen tuoteproteiinin määrä. Tätä menetelmää sovellettiin myös vierasproteiinin tuottoon metylotrofisella Pichia pastoris -hiivalla, jolloin saavutettiin korkeammat solutiheydet ja suurempi aktiivisen tuoteproteiinin määrä. Glukoosin määrä kasvatusliuoksessa pysyi riittävän alhaisena, jotta se ei repressoinut hiivan alkoholioksidaasi-promoottoria. Panossyöttömenetelmä on sopiva high-throughput-mikrobikasvatuksiin, koska sitä voidaan käyttää kuoppalevyillä ilman syöttölaitteita. Menetelmää voidaan hyödyntää uusien bioteknisten tuotteiden kehittämisessä erityisesti silloin, kun tuottoisäntä on herkkä kasvuolosuhteiden suhteen ja mikrobin kasvua halutaan hallita.

Protein Tyrosine Phosphatases (PTPs) initiate, modulate and terminate key cellular processes by dephosphorylating phosphotyrosine (pY) residues on signaling proteins. The coordinated action of PTPs with their cognate tyrosine kinases is crucial for the maintenance of cellular homeostasis. Five Receptor Tyrosine Phosphatases (RPTPs) DLAR, PTP99A, PTP69D,PTP10D and PTP52F are involved in the axon guidance process of the fruit-fly Drosophila melanogaster. The receptors in these RPTPs comprise of Cell Adhesion Molecules (CAMs) whilethe cytosolic region contains the catalytic PTP domains. Extensive studies on the genetic interactions between these RPTPs reveal that these five RPTPs collaborate, compete or are partially redundant in some developmental contexts. While the genetic interactions between these RPTPs are well characterized, the role of domain-domain interactions and the mechanism(s) of substrate recognition are poorly understood. The aim of this study was to understand the molecular basis for these interactions using a combination of biophysical, biochemical and structural biology tools. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis highlights the mechanistic issues in signal transduction with an emphasis on the role of the RPTPs in the neuro-development of Drosophila melanogaster. The first part of this chapter describes the structural features and the catalytic mechanism of the PTP domain. This is followed by a description of the mechanisms that modulate the activity of a PTP domain. The latter part of the chapter summarizes the role ofthese RPTPs in axon guidance of Drosophila melanogaster. The interactions between the RPTPsbased on genetic data provide a mechanistic hypothesis that could be examined in vitro. The studies described in the subsequent chapters of this thesis were performed to evaluate this hypothesis. Chapter 2: This chapter reports our observations on the so-called construct dependence on the expression of recombinant PTP domains in Escherichia coli. This chapter details the strategies used to obtain recombinant PTP domains in a soluble form suitable for biochemical and structural studies. This study involved substantial optimization in the size of the protein and overexpression strategies to avoid inclusion-body formation. Five strains of E. coli as well as three variations in purification tags viz., poly-histidine peptide attachments at the N-and C-termini and a construct with Glutathione-S-transferase at the N-terminus were examined. In this study, we observed that inclusion of a 45 residue stretch at the N-terminus was crucial for the over-expression of the PTP domains, influencing both the solubility and the stability of these recombinant proteins. While the addition of negatively charged residues in the N-terminal extension could partially rationalize the improvement in the solubility of these constructs, conventional parameters like the proportion of order-promoting residues or the aliphatic index did not correlate with the improved biochemical characteristics. The findings in this chapter suggest that the inclusion of additional parameters like secondary structure propensities apart from rigid domain predictions could play a crucial role in obtaining a soluble recombinant protein upon expression in E. coli. Chapter 3: This chapter reports the crystal structure of the PTP domain of PTP10D and PTP10Dsubstrate/inhibitor complexes. These structural studies revealed aromatic ring stacking interactions that mediate substrate recruitment into the PTP active site. In particular, these studies revealed the role of conserved aromatic residue in Motif 1 (Phenylalaline 76 in case ofPTP10D). Mutation of Phenylalanine 76 residue to a Leucine (similar to the mutation found in the inactive distal PTP domains in other bi-domain PTPs) resulted in a sixty-fold decrease in the catalytic efficiency of the enzyme. Fluorescence kinetic measurements to monitor ligand binding showed a three fold increase in the half time of enzyme-ligand complex formation. These studies highlight the role of the KNRY loop in substrate recruitment at the active of the PTP domain and the role of this segment in modulating the kinetics of the enzyme-substrate complex formation. Chapter 4: This chapter describes a strategy to utilize protein-protein interaction data to identify putative peptide substrates for a given protein. This study was performed in collaboration with Shameer Khader and Prof. R. Sowdhamini at the National Center for Biological Sciences (NCBS).This integrated search approach, called ‘PeptideMine’ was developed into a web-server for experimental and computational biologists. The Peptide Mine strategy combines sequence searches in the 'interacting sequence space' of a protein using sequence patterns or functional motifs. A compilation of indices that describe the chemical and solubility properties of potential peptide substrates to facilitate investigation by in vitro or in silico studies is also obtained from this server. The biological significance of such a design-strategy was examined in the context of protein-peptide interactions in the case of RPTPs of Drosophila melanogaster. Chapter 5: In this chapter, we report an analysis of the influence of the membrane distal (D2) domain on the catalytic activity and substrate specificity of the membrane proximal (D1) domain using two bi-domain RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of the Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A revealed a conformational rationale for the experimental observations. These studies suggested that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain. Chapter 6: This chapter describes biochemical studies to understand the role of the D2 domain of PTP99A. While the catalytic activity of PTP99A is localized to its membrane proximal (D1)domain, the inactive membrane distal (D2) domain influences the catalytic activity of the D1domain. Phosphatase activity, monitored using small molecule as well as peptide substrates, suggested that the D2 domain activates D1. Thermodynamic measurements on the bi-domain(D1-D2 protein) as well as single domain PTP99A protein constructs suggest that the presence of the inactive D2 domain influences the stability of the bi-domain protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by a few interactions at the inter-domain interface. In particular, we note that mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent position to the so-called allosteric site of a canonical PTP, PTP1B. These observations suggest functional optimization in bi-domain RPTPs wherein the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme. Chapter 7: This chapter summarizes the experimental and computational studies on the Drosophila melanogaster PTP domains. The salient features of the experimental data that revealed hitherto uncharacterized sequence-structure relationships in the conserved PTP domain are highlighted. The latter part of this chapter briefly suggests the scope of future research in this area based on some of the findings reported in this thesis. Appendix : This thesis has an appendix section with four parts. These comprise of technical details and auxiliary work that was not included in the main text of the thesis. Appendix I describes cloning strategies, purification protocols and a list of all recombinant proteins used in this study. Appendix II describes the standardization of the ‘Three Phase partitioning’ protocol for refolding and solubilization of protein from inclusion bodies. Appendix III includes theimmunochemical work performed to elucidate the localization of PTP10D in Drosophila embryos. Appendix IV describes the work on a Quercetin 2,3 Dioxygenase from Bacillus subtilis with an emphasis on the role of metal ions in modulating catalytic activity in this class of proteins.

La chromatographie mode mixte représente l’une des plus grandes évolutions de ces dernières années dans le domaine des bioséparations. Cette technique repose sur l'intervention de plusieurs types d'interactions au sein d'un seul et même support. Les résines de chromatographie mode mixte HEA, PPA et MEP HyperCel portent des groupements aliphatiques, aromatiques, thiophiliques ainsi que des groupements aminés protonables en différentes positions. Au moyen d’expériences de chromatographie, à l’aide de protéines standards aux propriétés spécifiques et de mélanges complexes, nous avons isolé ces différentes interactions. Nous avons mis en évidence l’intervention majeure d'au moins deux types d'interactions au sein de ces supports : interactions hydrophobes et électrostatiques. Nous avons pu observer le comportement des résines lors de variations de pH, de force ionique, de types de sels et de tampons ou lors de la présence d'autres composés organiques. Nous avons mis en évidence l'intervention combinée de ces types d'interactions lors des différentes phases de chromatographie. Le comportement des résines mode mixte a révélé des sélectivités particulières et dont le contrôle ciblé à l'aide de l'environnement a permis le développement de méthodes de purification efficaces et originales. Nous avons pu ainsi développer des applications telles que la purification de fragment d’anticorps (Fab’2) à partir de culture de cellules d’insectes, la capture de protéine de type MBP à partir d’extrait bactériens et la capture d’anticorps monoclonaux à partir de cellules de mammifères (CHO), et ainsi améliorer les conditions d’utilisation de la chromatographie en mode mixte. / Mixed mode chromatography is the most innovative technique for bioseparation. Mixed mode resins, as the term suggest, involves multiples types of interaction at the same time. HyperCel mixed mode resins, HEA, PPA and MEP, involve aliphatic, aromatic or thiophilic groups as well as protonable amine located in the spacer arm or as a head group. Using classical chromatographic experiments, standards proteins and complex mixtures, we highlighted the two major types of interactions involved: hydrophobic and electrostatic interactions. We specifically influenced these interactions by modifying the environment in terms of ionic strength, pH, salt types, and other compounds. The combination of these interactions during every phase of a chromatographic process has been demonstrated. Mixed mode resins thus offer unique selectivity that can be controlled by the environment. This allowed us to develop several applications from antibodies fragments capture from insect cells, to the purification of MBP-tagged proteins, through monoclonal antibody capture from CHO cells. We thus enhanced mixed mode chromatography.

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

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-β (Transforming Growth Factor β ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto \"in vitro\" como \"in vivo,\" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação \"in vitro\" e formação de osso \"in vivo\", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil. / The BMPs (Bone Morphogenetic Proteins) are members of the TGF-β (Transforming Growth Factor β) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction \"in vitro\" and \"in vivo\" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation \"in vitro\" and bone formation \"in vivo\" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil.

A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%). / The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). Cation exchange was chosen for the last chromatography. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%).

Uma abordagem promissora para a obtenção de coquetéis enzimáticos eficientes para a etapa de hidrólise na produção de etanol de 2ª geração é o enriquecimento em termos de atividades que lhes faltam, mas podem ser obtidas através de sistemas heterólogos. O trabalho teve como objetivo avaliar o sistema toxina -antitoxina (TA) para estabilização plasmidial em E. coli na produção de expansina e endoglucanase recombinantes. Os resultados indicaram que o sistema de expressão com estabilização TA é tão eficaz quanto o dependente de antibiótico para estabilização plasmidial. Além disso, mostrou-se mais eficiente pelo fato de não permitir a sobrevivência de células sem o plasmídeo. Estudos indicaram a quantidade mínima de 0,05mM de IPTG para expressão das proteínas nesta linhagem, cerca de 20 vezes menos que a concentração usualmente aplicada no momento da indução. Sendo assim, o sistema de estabilização plasmidial TA mostrou-se uma ótima ferramenta para o desenvolvimento de uma plataforma alternativa para a produção de proteínas recombinantes. / A promising approach to obtain efficient enzyme cocktails for the hydrolysis step in the production of 2nd generation ethanol is the enrichment in terms of activities that are lacking, but can be obtained by heterologous systems. The study aimed to evaluate the toxina-antitoxin (TA) system for plasmid stabilization in E. coli in the production of recombinant expansin and endoglucanase. The results indicated that the expression system with TA stabilization is as effective as the antibiotic dependent for plasmid stabilization. Moreover, it proved to be more efficient by not allo wing the survival of cells without the plasmid. Studies have indicated the minimum amount of 0.05 mM IPTG for expression of proteins in this strain, about 20 times less than the concentration usually applied for induction. Thus, the plasmid stabilization TA system proved to be a great tool for the development of an alternative platform for producing recombinant proteins.