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Expression and purification of ATP sulfurylase from Saccharomyces cerevisias in Escherichia coli and its application in pyrosequencing / 生物工程学报
Chinese Journal of Biotechnology ; (12): 623-627, 2007.
Article em Zh | WPRIM | ID: wpr-327976
Biblioteca responsável: WPRO
ABSTRACT
ATP sulfurylase (ATPS,EC 2.7.7.4) reversibly catalyzes the reaction between ATP and sulfate to produce APS and pyrophosphate (PPi), and has been used in pyrosequencing. The gene coding ATP sulfurylase was amplified from the genomic DNA of Saccharomyces cerevisias (CICC 1202), and cloned into prokaryotic expression plasmid pET28a( + ) to provide a recombinant expression plasmid pET28a( + )-ATPS. Upon IPTG induction, ATP sulfurylase was produced by E. coli BL21 (DE3) harboring the recombinant expression plasmid pET28a( + )-ATPS. The relative molecular weight of recombinant ATP sulfurylase with His tag was about 60 kD. The recombinant ATP sulfurylase with electrophoretic pure grade was obtained only by two purification steps: His * Bind Resin affinity chromatography and ultrafiltration. The specific activity of the purified recombinant ATP sulfurylase was as high as 5.1 x 10(4) u/mg. The successful application of the enzyme in pyrosequencing was also demostrated.
Assuntos
Texto completo: 1 Base de dados: WPRIM Assunto principal: Saccharomyces cerevisiae / Sulfato Adenililtransferase / Proteínas Recombinantes / Proteínas Fúngicas / Reação em Cadeia da Polimerase / Análise de Sequência de DNA / Escherichia coli / Genética / Metabolismo / Métodos Idioma: Zh Revista: Chinese Journal of Biotechnology Ano de publicação: 2007 Tipo de documento: Article
Texto completo: 1 Base de dados: WPRIM Assunto principal: Saccharomyces cerevisiae / Sulfato Adenililtransferase / Proteínas Recombinantes / Proteínas Fúngicas / Reação em Cadeia da Polimerase / Análise de Sequência de DNA / Escherichia coli / Genética / Metabolismo / Métodos Idioma: Zh Revista: Chinese Journal of Biotechnology Ano de publicação: 2007 Tipo de documento: Article