RESUMO
A procedure was established for the rapid isolation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (PEPCK) from an overproducing strain. Overexpression was achieved by the transformation of yeast cells with the multicopy plasmid YEp352 harbouring the PEPCK structural gene. The enzyme was purified to homogeneity using first anion-exchange chromatography on Q-Sepharose followed by hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration on Sephacryl S200. The purified phosphoenolpyruvate carboxykinase was further characterized with respect to the molecular mass, displaying an apparent molecular mass corresponding to a tetrameric form.
Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Fosfoenolpiruvato Carboxiquinase (ATP)/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Eletroforese em Gel de PoliacrilamidaRESUMO
The yeast hexokinase isoenzymes PI and PII have been purified in large amounts (20 mg) from overproducing yeast strains. The purification procedures of hexokinase PI and PII include anion-exchange chromatography on DEAE-Sephacel and chromatofocusing on PBE 94, hydrophobic interaction chromatography on phenyl-Sepharose (necessary for the isolation of the isoenzyme PI); in the final step either a Mono Q HR 5/5 or a Fractogel EMD TMAE 650(S) column was used. Hexokinase preparations were characterized before crystallization by chromatofocusing on a Mono P HR 5/20 FPLC column, where different forms of hexokinase can be rapidly distinguished by their elution behaviour. From both purified hexokinase PI and PII, large crystals were grown that diffract X-rays to high resolution.
