RESUMO
We have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Ribosomes interact with an alkyl linker attached to the resin, apparently through their RNA component. Examples are given with ribosomes from Escherichia coli, Deinococcus radiodurans, and with clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). The ribosomes obtained by this method are unusually intact, so that highly active ribosomes can now be isolated from the clinical isolates, enabling significantly improved in vitro functional assays that will greatly assist the discovery and development of new ribosomally targeted antibiotics.
Assuntos
Bactérias/ultraestrutura , Cromatografia Líquida/métodos , Ribossomos , Bactérias/patogenicidade , Espectrometria de Massas , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificaçãoRESUMO
A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.
Assuntos
Proteínas de Bactérias/química , Deinococcus/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores de Bactérias/química , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Fracionamento Celular , Cristalografia por Raios X , Deinococcus/genética , Deinococcus/crescimento & desenvolvimento , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Subunidades Ribossômicas Maiores de Bactérias/genéticaRESUMO
We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.