The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.

O Ácido Hialurônico, HA, é um dos principais componentes da matriz extracelular dos vertebrados. É um glicosaminoglicano hidrolisado pelas enzimas da família da Hialuronidase, envolvidas na regulação de importantes processos biológicos, como a permeabilidade vascular e a angiogênese. Conforme o interesse pelo desenvolvimento de uma rota de síntese para esta enzima, objetivamos a obtenção de um plasmídeo, contendo a sequência que codifica a variante 8 do gene Hyal-1. Para isto, planejou-se a inserção de dois sítios de restrição para a sub-clonagem, sítio dirigida, BamH-1 na região 5\' e Not-1 na 3\' na sequência do códon da Hyal-1. O inserto foi sub-clonado no plasmídeo, pET28-a, e transfectado para expressão em Escherichia coli Bl-21. A expressão foi induzida por IPTG no melhor tempo, de 4 horas, e a confirmação da expressão protéica foi realizada por Western blotting. Observou-se uma proteína de 45 kDa, confirmando a presença da Hyal-1. Realizou-se a expressão em 4 litros de cultura para obtenção de uma quantia suficiente para purificação em coluna de níquel-agarose. O rendimento neste experimento foi de 25µg por litro. A rota sintética sugerida neste estudo mostrou-se eficiente para obtenção da proteína Hyal-1 recombinante, justificando futuros investimentos na otimização deste processo / Hyaluronic Acid, HA is a major component of the extracellular matrix of vertebrates. It is a glycosaminoglycan hydrolyzed by enzymes of the hyaluronidase family, involved in the regulation of important biological processes such as angiogenesis and vascular permeability. As interest in the development of a synthesis route for this enzyme, we aim to obtain a plasmid containing the coding sequence of the gene variant 8 Hyal-1. To obtain the plasmid insert was planned and two restriction sites for sub-cloning site directed at the 5\' Bam H-1 and 3\' Not-1 in codon sequence of Hyal-1. The insert was sub-cloned into plasmid pET28-a, and transfected for expression in Escherichia coli Bl-21. The expression was induced by IPTG in the best time of 4 hours and confirmation of protein expression was performed by Western blotting. There was a 45 kDa protein, thus confirming the presence of Hyal-1. Purification was performed on nickel-agarose column to obtain a larger amount of the protein, approximately 25µg/L. The route suggested in this study was efficient attainment of Hyal-1 recombinant protein.

O Ácido Hialurônico, HA, é um dos principais componentes da matriz extracelular dos vertebrados. É um glicosaminoglicano hidrolisado pelas enzimas da família da Hialuronidase, envolvidas na regulação de importantes processos biológicos, como a permeabilidade vascular e a angiogênese. Conforme o interesse pelo desenvolvimento de uma rota de síntese para esta enzima, objetivamos a obtenção de um plasmídeo, contendo a sequência que codifica a variante 8 do gene Hyal-1. Para isto, planejou-se a inserção de dois sítios de restrição para a sub-clonagem, sítio dirigida, BamH-1 na região 5\' e Not-1 na 3\' na sequência do códon da Hyal-1. O inserto foi sub-clonado no plasmídeo, pET28-a, e transfectado para expressão em Escherichia coli Bl-21. A expressão foi induzida por IPTG no melhor tempo, de 4 horas, e a confirmação da expressão protéica foi realizada por Western blotting. Observou-se uma proteína de 45 kDa, confirmando a presença da Hyal-1. Realizou-se a expressão em 4 litros de cultura para obtenção de uma quantia suficiente para purificação em coluna de níquel-agarose. O rendimento neste experimento foi de 25µg por litro. A rota sintética sugerida neste estudo mostrou-se eficiente para obtenção da proteína Hyal-1 recombinante, justificando futuros investimentos na otimização deste processo / Hyaluronic Acid, HA is a major component of the extracellular matrix of vertebrates. It is a glycosaminoglycan hydrolyzed by enzymes of the hyaluronidase family, involved in the regulation of important biological processes such as angiogenesis and vascular permeability. As interest in the development of a synthesis route for this enzyme, we aim to obtain a plasmid containing the coding sequence of the gene variant 8 Hyal-1. To obtain the plasmid insert was planned and two restriction sites for sub-cloning site directed at the 5\' Bam H-1 and 3\' Not-1 in codon sequence of Hyal-1. The insert was sub-cloned into plasmid pET28-a, and transfected for expression in Escherichia coli Bl-21. The expression was induced by IPTG in the best time of 4 hours and confirmation of protein expression was performed by Western blotting. There was a 45 kDa protein, thus confirming the presence of Hyal-1. Purification was performed on nickel-agarose column to obtain a larger amount of the protein, approximately 25µg/L. The route suggested in this study was efficient attainment of Hyal-1 recombinant protein.

<p>Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.</p>

Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.