| Summary: | 碩士 === 國立彰化師範大學 === 生物技術研究所 === 97 === Aphids are a well known pest insect in agriculture. They have lots of endosymbiont, including the primary endosymbiont Buchnera. The relationship between Buchnera and aphids is obligatively mutualistic; Buchnera supplies essential amino acids to aphids, and aphids supply nonessential amino acids to Buchnera in return. To study the enzymes of amino acid biosynthesis pathway can not only reveal the co-evolution between Buchnera and aphids but also make these enzymes pesticide targets to control aphids. In this experiment, we studied the biochemical characteristics of 3-deoxy-D-arabino-2-heptulosonate 7-phosphate synthetase (DAHPS EC 2.5.1.54), the first enzyme of the shikimate pathway. BaDAHPS was expressed in E.coli BL21(DE3) cells. The enzyme was precipitated with 50-75% saturation of ammonium sulfate, and purified by Ni-chelating affinity column.The enzyme expressed by pET20b(+) was mostly inclusion bodies, only few DAHPS were native form, make it hard to purify DAHPS.
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