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首页> 外文期刊>Advances in Natural Sciences >EXPRESSION AND CHARACTERIZATION OF GLYCININ proA_5A_4B_3 AS A HIGHLY SPECIFIC SUBSTRATE FOR SOYBEAN MATURATION ENZYME
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EXPRESSION AND CHARACTERIZATION OF GLYCININ proA_5A_4B_3 AS A HIGHLY SPECIFIC SUBSTRATE FOR SOYBEAN MATURATION ENZYME

机译：大豆成熟酶高特异性底物甘草素proA_5A_4B_3的表达与表征。

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摘要

The expression vector for glycinin proA_5A_4B_3 was constructed by using pET-21a(+) vector (Novagen) and the original cDNA plasmid pGA_5A_4B_3-822 as starting material. The resulting recombinant plasmids, designated as pEA_5A_4B_3-5 and pEA_5 A_4B_3-7 were used to express the glycinin proA_5A_4B_3 in E. coli strain BL21 (DE3). The expressed protein assembled in oligomers, trimers and hexamers, was purified to homogeneity and served as a highly specific substrate for soybean maturation enzyme isolated from commercial fresh raw flour of soybean mature seeds. The SDS/PAGE patterns for the time-course digestion study showed that the maturation enzyme specifically hydrolyzed first the bond Asn~(355)-Gly to release the basic (B_3) and acidic (A_5A_4) subunits. The following cleavage site was the bond Asn~(97)-Arg between A_4 and A_5 subunits. This specific stepwise cleavage was confirmed by kinetics for cleavage of the unmodified and/or modified sequences represented by the peptides containing the amino acids found at two processing sites of glycinin proA_5A_4B_3, Cys-Glu-Thr-Arg-Asn~(355)-Gly-Val-Glu (A_4-B_3) and Gln-Glu-Gln-Ser-Asn~(97)-Arg-Arg-Gly (A_5-A_4).
机译：用pET-21a（+）载体（Novagen）和原始cDNA质粒pGA_5A_4B_3-822作为起始材料，构建了大豆球蛋白proA_5A_4B_3的表达载体。所得的重组质粒，分别称为pEA_5A_4B_3-5和pEA_5A_4B_3-7，用于在大肠杆菌BL21（DE3）菌株中表达大豆球蛋白proA_5A_4B_3。将表达的蛋白组装在低聚物，三聚体和六聚体中，纯化至均一，并用作从大豆成熟种子的商业新鲜生面粉中分离出来的大豆成熟酶的高特异性底物。用于时程消化研究的SDS / PAGE模式显示，成熟酶首先特异水解Asn〜（355）-Gly键以释放碱性（B_3）和酸性（A_5A_4）亚基。接下来的切割位点是A_4和A_5亚基之间的Asn〜（97）-Arg键。这种特异性的逐步切割通过动力学的未切割和/或经修饰的序列的切割来证实，所述肽由在大豆球蛋白proA_5A_4B_3，Cys-Glu-Thr-Arg-Asn〜（355）-Gly的两个加工位点发现的氨基酸所代表的肽代表-Val-Glu（A_4-B_3）和Gln-Glu-Gln-Ser-Asn〜（97）-Arg-Arg-Gly（A_5-A_4）。

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