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Expression, Purification and Activity Identification of Arabidopsis thaliana Alpha-dioxygenase 2 (AtDOX2)in Pichia pastoris
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Abstract  In order to obtain active Arabidopsis thaliana alpha-dioxygenase 2 (AtDOX2), the encoding sequence of AtDOX2 was cloned into pPIC9k to obtain pPIC9k-AtDOX2. The recombinant vector was linearized and electrophorated into Pichia pastoris strain GS115. After G418 selection, PCR analysis and optimization of methanol inducing time,the high level expression strain of GS115/pPIC9k-AtDOX2 was obtained. SDS-PAGE analysis revealed that the expression level of recombinant protein reached to top at 96 h after inducing by 0.5% methanol and the target protein accounted for 15% of the extracellular total protein. The 70 kD recombinant AtDOX2 was purified by the Ni-NTA chromatography column, and the purity of recombinant protein was more than 80%. The peroxidase activity of the purified protein was tested by the 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) assay, the result showed that the recombinant protein had peroxidase activity, and the peroxidase activity of AtDOX2 could be activated by Ca2+ and Mg2+ and inhibited by EDTA, imidazole and Mn2+. The α-dioxygenase activity was analyzed by 2,4-dinitrophenyl hydraz(2, 4-DNP) assay, the result showed that the recombinant protein had dioxygenase activity, and the α-dioxygenase activity of AtDOX2 could be activated by Ca2+ too. Results indicate that the ecombinant AtDOX2 was expressed and purified by yeast system, and it acts as a dual functional enzyme of peroxidase and dioxygenase.
Key wordsArabidopsis thaliana      AtDOX2      Pichia pastoris      Peroxidase      α-dioxygenase     
Received: 04 February 2010     
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NULL. Expression, Purification and Activity Identification of Arabidopsis thaliana Alpha-dioxygenase 2 (AtDOX2)in Pichia pastoris[J]. , 2011, 19(1): 51-56.
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http://journal05.magtech.org.cn/Jwk_ny/EN/     OR     http://journal05.magtech.org.cn/Jwk_ny/EN/Y2011/V19/I1/51
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