Molecular Cloning of a cDNA Encoding Granule-bound Starch Synthase I from Kidney B Bean Seeds and Expression in Escherichia coil
Molecular Cloning of a cDNA Encoding Granule-bound Starch Synthase I from Kidney B Bean Seeds and Expression in Escherichia coil
复制标题
肾 B 豆种子颗粒结合淀粉合酶 I cDNA 的分子克隆及其在大肠杆菌中的表达
DOI：
10.5458/jag.50.355
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发表时间：
2003
影响因子：
1.1
通讯作者：
H. Matsui
中科院分区：
文献类型：
--
作者：
N. Isono;Hiroyuki Ito;T. Senoura;Motoyo Yoshikawa;K. Nozaki;H. Matsui
芸豆（Phaseolus vulgaris L .）种子淀粉颗粒的主要相关蛋白是一种颗粒结合淀粉合成酶I（标记为PvGBSSI），分子量为60 kDa。为了分析天然颗粒结合淀粉合成酶I （GBSSI）酶的性质，而不是从淀粉颗粒中溶解的酶，我们进行了一项研究，确定了在种子发育过程中表达的GBSSI序列，并表征了编码的重组酶的酶学性质。从芸豆未成熟种子cDNA文库中分离到一个cDNA克隆（命名为pvgbssl）。预测的成熟PvGBSSI初级序列与其他GBSSI成员具有显著的同一性（66% - 81%）。Northern blot和淀粉颗粒蛋白分析显示，PvGBSSI转录物和蛋白在种子成熟后期达到最高水平。为了研究重组PvGBSSI （rPvGBSSI）的酶学性质，利用sds -聚丙烯酰胺凝胶一步柱层析法从埃希氏菌线圈中纯化出单带蛋白。在0.25 M柠檬酸盐的作用下，rPvGBSSI的活性被刺激了1.7倍。添加0.25 M柠檬酸时支链淀粉的亲和力明显高于不添加柠檬酸时的亲和力，且添加或不添加柠檬酸时的最大速度不变。
The major protein associated with starch granules from kidney bean (Phaseolus vulgaris L .) seeds is a granule-bound starch synthase I (designated PvGBSSI) with the molecular mass of 60 kDa. To analyze the nature of native granule-bound starch synthase I (GBSSI) enzyme, not the enzymes solubilized from starch granules, a study was undertaken to identify the GBSSI sequence expressed during seed development and to characterize the enzymatic properties of the coded recombinant enzyme . A cDNA clone (designated pvgbssl) was isolated from a cDNA library of kidney bean immature seeds . The predicted primary sequence of mature PvGBSSI displayed significant identity (66-81 %) to those of other GBSSI members . Analyses of Northern blot and starch granule proteins revealed that both transcript and protein for PvGBSSI showed maximum levels at the late to mature stages of seed maturation . To investigate enzymatic properties, recombinant PvGBSSI (rPvGBSSI) was purified from Escherichia coil as a single band of protein on SDS-polyacrylamide gel by one-step column chromatography. The activity of rPvGBSSI was stimulated 1.7-fold in the presence of 0.25 M citrate. The affinity for amylopectin with 0.25 M citrate was much higher than for that without citrate, while the maxim um velocities were constant with or without citrate .