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| Expression,purification and subcellular prediction of Arabidopsis alpha-Dioxygenase 2 |
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Abstract Alpha-Dioxygenase 2 is obtained from ABRC,the coding sequence is amplified using primers containing SmaI and XhoI digest cites,then inserted into pMD-T simple vector,make sure it is right by sequencing,DOX2 is inserted into pGEX-5X-1 by double digest and ligation,then it is transformed into BL21 （DE3） pLysS and BL21 (DE3)-RIPL codon +,DOX2 is expressed efficiently in BL21 (DE3)-RIPL codon + by SDS-PAGE and Western blot analysis,further solubility analysis indicates most of the recombinant protein is expressed in the form of inclusion body,soluble part is harvested by GST affinity purification,and it is used to activity test.Bioinformatics analyisis indicates: DOX2 gene has 88 rare codons in E.coli,nearly 14% of the total codons.Subcellular prediction by SVM indicates:the enzyme is likely to locate in cytoplast.
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Received: 04 January 2007
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