ProduÃÃo e purificaÃÃo de DNA plasmidial a partir de Escherichia coli recombinante

AUTOR(ES)

Keila Aparecida Moreira

DATA DE PUBLICAÇÃO

2003

RESUMO

The production and purification of plasmid DNA with high degree of purity have been having a large interest in the last years, mainly due to the development of several techniques, such as molecular medicine, gene therapy and DNA vaccination. The different bacterial growth conditions were set up in order to analyze the plasmid pD2 production, such as the influence of agitation velocities (120, 160 and 200 rpm), kanamycin concentrations (10, 20, 30, 40 and 50mg ml-1) and culture medium (Terrific Broth - TB or Luria Bertani with glucose - LBG). Were also investigated the effect of plasmid DNA, RNA and total proteins in the aqueous two phases systems (ATPS), polyethylene glycol (PEG)/ sodium phosphate salt (K2HPO4) on the plasmid (pD2). Was also analyzed the influence of the molecular weight of PEG, load of lyse solution (20, 40 or 60% w/w total mass of the systems) on the plasmid DNA partition. With the pVax-LacZ plasmid the precipitation with ammonium sulphate (2.0, 2.5 and 3.0M) followed by hydrophobic interaction chromatography (HIC) using as support Phenyl Sepharose 6 Fast Flow was applied. The agitation velocity that presented better results related to biomass and plasmid DNA production was 200 rpm, while 30-50mg mL-1 of kanamycin presented similar results for all the studied conditions. The TB medium was the best medium for biomass and DNA production. The plasmid was stable in TB medium, showing 82% plasmid containing cells. The plasmid DNA are partitioned between the top an bottom phases showing to be dependent to the molecular weight of the system and a good amount of cell lysate protein was accumulated in the interphase of the system. The best recovery plasmid yield (37%) was obtained with PEG 400 system with a 60% (w/w) lysate load. The precipitation procedure with ammonium sulphate (2.5M) followed by the hydrophobic interaction chromatography were capable to separate proteins and genomic DNA of the plasmid, obtaining 51% of revenue and a purification factor of 3.3 after the precipitation with ammonium sulphate

ASSUNTO(S)

escherichia coli biotecnologia microbiologia ciencias biologicas plasmÃdeos bactÃrias

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