RESUMO
The fungal genus Pyrenochaetopsis has received particular attention because of its different lifestyles, such as numerous plant pathogenic, saprophytic, and endophytic species. Its ability to infect plant cells relies heavily upon secreted peptidases. Here, we investigated the biochemical properties and catalytic specificity of a new serine peptidase secreted by the filamentous fungus Pyrenochaetopsis sp. We found that while this neutral serine peptidase displayed optimal activity at a pH of 7.0 and temperature of 45 °C, it tolerated a wide range of pH conditions and temperatures lower than 45 °C. Its peptidase activity was depressed by some metallic ions (such as aluminum, cobalt, and copper (II) chloride) and enhanced by others (such as sodium, lithium, magnesium, potassium, calcium, and manganese). Lastly, the enzyme showed the greatest specificity for non-polar amino acids, particularly leucine and isoleucine, and moderate specificity for basic and neutral polar amino acids. It displayed the least specificity for acidic residues.
Assuntos
Ascomicetos/enzimologia , Biocatálise , Serina Proteases/química , Serina Proteases/metabolismo , Inibidores Enzimáticos/farmacologia , Guanidina/farmacologia , Metais/farmacologia , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Tensoativos/farmacologia , Temperatura , Ureia/farmacologiaRESUMO
Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line.
Assuntos
Fator IX/genética , Fator IX/metabolismo , Vírus da Leucemia Murina de Moloney/fisiologia , Vírus do Sarcoma Murino de Moloney/fisiologia , Integração Viral , Linhagem Celular , Vetores Genéticos , Humanos , Vírus da Leucemia Murina de Moloney/genética , Vírus do Sarcoma Murino de Moloney/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução GenéticaRESUMO
The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl(2), KCl, and BaCl, and partially inhibited by CuCl(2), CoCl(2) and totally inhibited by AlCl(3) and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k (cat)/K (m) of 10,666 mM(-1) s(-1), followed by the peptide Abz-GLRSSKQ-EDDnp with a k (cat)/K (m) of 7,500 mM(-1) s(-1). Basic and acidic side chain-containing amino acids performed best at subsite S(1). Subsites S(2), S(3), S(') (2), and S(') (1), S(') (3) showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k (cat)/K (m) were observed for the subsites S(2), S(3), and S(') (2.) The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Penicillium/enzimologia , Serina Proteases/química , Serina Proteases/isolamento & purificação , Sequência de Aminoácidos , Estabilidade Enzimática , Espaço Extracelular/química , Espaço Extracelular/enzimologia , Espaço Extracelular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Dados de Sequência Molecular , Penicillium/química , Penicillium/genética , Transporte Proteico , Serina Proteases/genética , Serina Proteases/metabolismo , Especificidade por SubstratoRESUMO
Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.
