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Expression, purification, and crystallization of a novel galactose mutarotase from Thermoanaerobacter tengcongensis / 中国医学科学院学报
Acta Academiae Medicinae Sinicae ; (6): 696-701, 2009.
Artigo em Chinês | WPRIM | ID: wpr-301624
ABSTRACT
<p><b>OBJECTIVE</b>To purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.</p><p><b>METHODS</b>The tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200).</p><p><b>RESULT</b>The purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained.</p><p><b>CONCLUSIONS</b>We successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.</p>
Assuntos
Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Proteínas de Bactérias / Transformação Bacteriana / Carboidratos Epimerases / Química / Clonagem Molecular / Cristalização / Thermoanaerobacter / Escherichia coli / Vetores Genéticos / Genética Idioma: Chinês Revista: Acta Academiae Medicinae Sinicae Ano de publicação: 2009 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Proteínas de Bactérias / Transformação Bacteriana / Carboidratos Epimerases / Química / Clonagem Molecular / Cristalização / Thermoanaerobacter / Escherichia coli / Vetores Genéticos / Genética Idioma: Chinês Revista: Acta Academiae Medicinae Sinicae Ano de publicação: 2009 Tipo de documento: Artigo