RESUMO
The use of high-throughput screening (HTS) has been successfully applied over the past years in downstream process development of therapeutic proteins. Different HTS applications have been introduced to speed up the development of purification processes for these proteins. In the light of these findings, studies were conducted to develop a controlled method of pipetting the right amount of resin into self-packed filter plates. In total, 13 plates were tested for their suitability in an HTS application. Based on these studies, the Seahorse plate was experimentally verified as the best filter plate. Suitable conditions for preparing a self-packed plate were established so that linear correlations between resin concentration (POROS 50HS) and UV adsorption (420 nm) were obtained. The accuracy of dispensing the slurry into a microtiter plate was controlled within 1% deviation under established conditions. Overall, self-packed filter plates are equipped to be used in the development of purification processes in HTS mode.
Assuntos
Filtração/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Proteínas/isolamento & purificação , Compostos Azo/química , Biotecnologia/instrumentação , Etanol/química , Filtração/métodos , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/químicaRESUMO
A strain from the group of black Aspergilli was analysed in detail to determine the species to which it belongs. A detailed analysis of morphology, RFLP patterns and metabolite profiles was carried out. In addition, a phylogenetic tree was constructed for the black Aspergilli using the ITS and the beta-tubulin sequences of the individual strains. The new species differs by its poor growth on glycerol and galacturonate and its unique extrolite profile consisting of aurasperone B, nigragillin, asperazine and kotanins. RFLP analysis using three genes as probes also resulted in a unique pattern. These data indicate that the strain was closely related but not identical to Aspergillus foetidus, Aspergillus niger and Aspergillus tubingensis. It was therefore designated as a novel species and named Aspergillus vadensis.
Assuntos
Aspergillus/classificação , Aspergillus/fisiologia , Aspergillus/citologia , Aspergillus/genética , Impressões Digitais de DNA , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , DNA Espaçador Ribossômico/isolamento & purificação , Proteínas Fúngicas/genética , Glicerol/metabolismo , Ácidos Hexurônicos/metabolismo , Indóis/análise , Dados de Sequência Molecular , Filogenia , Piperazinas/análise , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Tubulina (Proteína)/genéticaRESUMO
A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.
