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Efficient expression of recombinant protein was achieved in E. coli through the use of vectors capable of copy number amplification. Expression was regulated by the tryptophan promoter and plasmid copy number by the temperature sensitive Lambda Pg promoter. Reproducible expression of a variety of recombinant proteins was achieved in defined medium under fermentation conditions. In all cases, cell growth was strongly inhibited after amplification of both plasmid DNA and product expression. Plasmid copy number control was studied in continuous culture. Below 36°G, plasmid DNA was maintained at a low and constant level. At 37°G and 38°C however, plasmid DNA amplification was activated. In order to study the effect of physiological parameters on expression from these plasmids, the CAT protein was chosen as a model. The effect of specific growth rate on copy number amplification was studied in batch and continuous culture. Prior to induction, higher growth rates increased CAT expression. After induction however, clear trends were difficult to determine although growth rate clearly influenced the lag period before DNA amplification occurred. In continuous culture, a combination of reduced growth rate and overgrowth by the plasmid free cells caused transient product formation. Therefore to conduct extended experiments under inducing conditions, two stage continuous culture was evaluated. Cells grown at 34°C were fed continuously to a second fermenter held at 38°C. Stable production was achieved over a 160 hour period - sufficient time to perform reproducible experiments. In these experiments, specific growth rate in the second stage was determined to be a significant factor influencing high level GAT expression. In addition, the efficiency by which it was both transcibed and translated from the plasmid DNA could be altered. The residence time in the second stage played a secondary role - mainly influencing the point at which plasmid-free cells could overgrow the culture. Trends in plasmid stability were also studied. The development of more sensitive methods to detect plasmid instability revealed the gradual accumulation of plasmid-free cells in these cultures. This was due to overgrowth of these plasmid-free cells rather than plasmid loss due to segregational deficiencies. Higher rates of overgrowth were observed at greater product expression levels.

Thesis (Ph. D.)--Illinois State University, 2007. / Title from title page screen, viewed on July 16, 2008. Dissertation Committee: Radheshyam K. Jayaswal (chair), Brian J. Wilkinson, Anthony J. Otsuka, Wade A. Nichols, Laura A. Vogel. Includes bibliographical references and abstract. Also available in print.

Babesia bovis is a causative agent of bovine babesiosis and is transmitted by vector ticks, Rhipicephalus (Boophilus) spp. The disease has a high mortality rate in susceptible cattle, causing serious economic loss. At present, the only commercial vaccine is culture-based with limited availability. No effective molecular vaccine has been developed to date. Generating a vaccine with specific critical epitopes responsible for protection against B. bovis is critically important. Immunity against B. bovis requires both innate and adaptive responses, with antigen-specific CD4+ T cells essential to the latter through production of IFN-γ. Fourteen B. bovis proteins were selected as putative vaccine candidates and their full-length genes cloned for recombinant protein production intended for evaluating peripheral blood mononuclear cell IFN-γ secretion level from experimentally infected animals in ELISPOT. All proteins expressed in insoluble form (inclusion bodies) and could not be purified. B. bovis genes were then truncated to exclude signal peptide and transmembrane regions, then cloned and expressed using pET101/D-TOPO in Escherichia coli to obtain soluble, useable proteins. Only recombinant B. bovis MSA1, MSA2b and MSA2a1 proteins were successfully expressed in soluble form. These proteins induce invasion-blocking antibodies in immunized cattle, are hypothesized to elicit protection in susceptible animals, but were previously studied by others. Due to failure to produce new candidates to assay, the animal experiments were not performed. Instead, sera from field-infected cattle were assayed for reactivity against the MSA proteins by indirect immunofluorescent antibody (IFA) and western blot (WB) analysis. Field sera from South Texas (#41) and the Mexican Yucatan (#6, #9 and #11) along with positive and negative controls were tested. In IFA test, cattle #6, #9 and #41 were positive while #11 was negative. In WB, #41 and #6 reacted with the recombinant MSA proteins and with control B. bovis whole parasite lysate. However, both #11 and #9 had no signal in WB, although the latter was positive in IFA. Several theories may explain this phenomenon, such as the different preparation process of the antigen in the two tests, strain differences between sera and test antigens, or the different design and nature of each test.

Orientador: José Osmar Gaspar / Banca: Mário Tyago Murakami / Banca: Marinônio Lopes Cornélio / Resumo: No Brasil, foram descritas mais de dez viroses em feijoeiro, citando-se aquela causada pelo Southern bean mosaic virus (SBMV) do gênero Sobemovirus. Este vírus tem uma restrita gama de hospedeiras, confinada quase exclusivamente a espécies da família das leguminosas, sendo algumas de interesse econômico como o feijoeiro comum e a soja. O SBMV é a espécie tipo do gênero, possui partículas isométricas (28-30nm) contendo RNA genômico de 4-4,5kb com polaridade positiva e proteína capsidial com massa molecular de 29-30 kDa. O genoma do SBMV é constituído por quatro Open Reading Frames (ORFs) com sobreposição entre elas. A ORF 1 codifica uma possível proteína do movimento, a ORF 2 uma poliproteína, que por clivagem, origina três produtos funcionais: uma serino protease, uma proteína ligadora do genoma viral (VPg) e uma polimerase com atividade relacionada à replicação do RNA viral (RNA polimerase RNA-dependente, RpRd). Dentro da ORF 2 encontra-se a ORF 3, que codifica uma proteína de função desconhecida. A ORF 4 codifica a proteína capsidial, traduzida a partir de um RNA subgenômico. O presente trabalho consiste na expressão da proteína P1 do SBMV-SP em Escherichia coli, sua purificação e a posterior utilização para a produção de antissoro policlonal. As atividades realizadas compreenderam a purificação das partículas virais do SBMV isolado São Paulo (SBMV-SP) a partir de folhas infectadas de Phaseolus vulgaris 'Jalo', obtenção do RNA viral a partir de partículas purificadas, produção de cDNA utilizando primer anti-senso, amplificação do gene da proteína do movimento, purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, corte enzimático com as enzimas Bam HI e Hind III, subclonagem nos vetores de expressão... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In Brazil, more than ten bean viroses were described, including the one caused by Southern bean mosaic virus (SBMV) from genus Sobemovirus. This virus has a strict host range, confined almost exclusively to species from leguminosas family, and some of them have economic importance, like the common bean and soya. SBMV is the type species from this genus, has isometric particles (28-30nm) that contains genomic RNA of 4-4,5kb with positive polarity and a capsidal protein with molecular mass of 29-30kDa. SBMV genome consistis of four Open Reading Frames (ORFs) with overlap between them. ORF 1 encodes a possible movement protein, ORF 2 a poliprotein, which origin three functional products by clivage: a serin protease, a viral genomic biding protein (VPg) and a polimerase. Inside ORF 2 is situated the ORF 3, that encodes a protein with unknow function. ORF 4 encodes the capsidal protein, translated by a subgenomic RNA. The activities performed until this moment were the purification of SBMV isolated São Paulo(SBMV-SP) viral particles, obtention of viral RNA from SBMV purified partcles, cDNA production using anti-sense primer, amplification of the supposed gene of movement protein, purification of the amplified fragment and later biding on expression vector pGEM-T. The recombinat vector were transformed in Escherichia coli cells competent and than purified and digested with Bam HI and Hind III enzime. The released fragment was subcloned in pMAL and pET28a, which were used on competent cells for expression tests. Analysis of gel poliacrialamida showed the efficiency of tests of expression. Using pET28a was observed an expression of a protein of approximately 22 kDa (17 kDa relating to MP + 5 kDa relating to the tail of histidine) which was purified in resin Ni-NTA by affinitive chromatography.Using pMAL was observed an expression of a protein of approximately... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-22Bitstream added on 2014-06-13T19:55:51Z : No. of bitstreams: 1 mariutti_rb_me_sjrp.pdf: 594995 bytes, checksum: 89516139d0654c33b1221cfd31a27c6d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / No Brasil, foram descritas mais de dez viroses em feijoeiro, citando-se aquela causada pelo Southern bean mosaic virus (SBMV) do gênero Sobemovirus. Este vírus tem uma restrita gama de hospedeiras, confinada quase exclusivamente a espécies da família das leguminosas, sendo algumas de interesse econômico como o feijoeiro comum e a soja. O SBMV é a espécie tipo do gênero, possui partículas isométricas (28-30nm) contendo RNA genômico de 4-4,5kb com polaridade positiva e proteína capsidial com massa molecular de 29-30 kDa. O genoma do SBMV é constituído por quatro Open Reading Frames (ORFs) com sobreposição entre elas. A ORF 1 codifica uma possível proteína do movimento, a ORF 2 uma poliproteína, que por clivagem, origina três produtos funcionais: uma serino protease, uma proteína ligadora do genoma viral (VPg) e uma polimerase com atividade relacionada à replicação do RNA viral (RNA polimerase RNA-dependente, RpRd). Dentro da ORF 2 encontra-se a ORF 3, que codifica uma proteína de função desconhecida. A ORF 4 codifica a proteína capsidial, traduzida a partir de um RNA subgenômico. O presente trabalho consiste na expressão da proteína P1 do SBMV-SP em Escherichia coli, sua purificação e a posterior utilização para a produção de antissoro policlonal. As atividades realizadas compreenderam a purificação das partículas virais do SBMV isolado São Paulo (SBMV-SP) a partir de folhas infectadas de Phaseolus vulgaris ‘Jalo’, obtenção do RNA viral a partir de partículas purificadas, produção de cDNA utilizando primer anti-senso, amplificação do gene da proteína do movimento, purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, corte enzimático com as enzimas Bam HI e Hind III, subclonagem nos vetores de expressão... / In Brazil, more than ten bean viroses were described, including the one caused by Southern bean mosaic virus (SBMV) from genus Sobemovirus. This virus has a strict host range, confined almost exclusively to species from leguminosas family, and some of them have economic importance, like the common bean and soya. SBMV is the type species from this genus, has isometric particles (28-30nm) that contains genomic RNA of 4-4,5kb with positive polarity and a capsidal protein with molecular mass of 29-30kDa. SBMV genome consistis of four Open Reading Frames (ORFs) with overlap between them. ORF 1 encodes a possible movement protein, ORF 2 a poliprotein, which origin three functional products by clivage: a serin protease, a viral genomic biding protein (VPg) and a polimerase. Inside ORF 2 is situated the ORF 3, that encodes a protein with unknow function. ORF 4 encodes the capsidal protein, translated by a subgenomic RNA. The activities performed until this moment were the purification of SBMV isolated São Paulo(SBMV-SP) viral particles, obtention of viral RNA from SBMV purified partcles, cDNA production using anti-sense primer, amplification of the supposed gene of movement protein, purification of the amplified fragment and later biding on expression vector pGEM-T. The recombinat vector were transformed in Escherichia coli cells competent and than purified and digested with Bam HI and Hind III enzime. The released fragment was subcloned in pMAL and pET28a, which were used on competent cells for expression tests. Analysis of gel poliacrialamida showed the efficiency of tests of expression. Using pET28a was observed an expression of a protein of approximately 22 kDa (17 kDa relating to MP + 5 kDa relating to the tail of histidine) which was purified in resin Ni-NTA by affinitive chromatography.Using pMAL was observed an expression of a protein of approximately... (Complete abstract click electronic access below)

<p>In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in <i>Escherichia coli</i>.</p><p>The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon.</p><p>The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR.</p><p>The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in <i>Escherichia coli</i>.</p>

In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in Escherichia coli. The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon. The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR. The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in Escherichia coli.

NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...

6 Abstract Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activity. This antitumor agent binds to DNA and forms covalent DNA adducts. Enzymes, which are involved in its enzymatic activation, are cytochromes P450 (CYP) and peroxidases. To elucidate the effect of ellipticine on the expression and enzymatic activity of the individual components of the microsomal mixed function oxidase system in different tissues, we used rat model. Simultaneously, the effect of ellipticine and its cytotoxicity on different tumor cell lines was also investigated. Another part of the presented work was targeted on preparation of anti-peptide antibody against orphan cytochrome P450 2S1, which is highly expressed in many human tumours of the epithelial origin, for its detection in these tissues. For better understanding how CYP2S1 can contribute to the metabolism of xenobiotics, the protein was prepared by heterologous expression in E. coli. Furher, its role in metabolism of an antitumor drug ellipticine, a carcinogenic environmental pollutant benzo[a]pyrene (BaP) and its derivate BaP-7,8-dihydrodiol was examined. Utilizing a mouse model, the impact of pulmonary inflammation on the metabolism of an environmental carcinogen was...