Mazorra-Manzano Miguel A, Yada Rickey Y
Department of Food Science, University of Guelph, Ontario, Canada N1G 2W1.
Phytochemistry. 2008 Oct;69(13):2439-48. doi: 10.1016/j.phytochem.2008.07.012. Epub 2008 Sep 14.
The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47kDa consisting of heavy and light chain components, approx. 32 and 16kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K(m) of 44 microM, and k(cat) of 55 s(-1) using a synthetic substrate. The enzyme was inhibited by pepstatin A.
本研究报道了来自拟南芥的一种典型天冬氨酸蛋白酶（AtAP A1）的重组表达、纯化及部分特性。编码AtAP A1前体的cDNA利用酵母毕赤酵母表达为一种功能性蛋白。发现重组AtAP A1的成熟形式是一种异源二聚体糖基化蛋白，分子量为47kDa，由重链和轻链组分组成，分别约为32kDa和16kDa，通过二硫键相连。糖基化通过轻链中的植物特异性插入片段发生。重组AtAP A1的催化特性与其他植物天冬氨酸蛋白酶相似，在酸性pH范围内具有活性，在pH 4.0时活性最高，使用合成底物时Km为44μM，kcat为55 s-1。该酶被胃蛋白酶抑制剂A抑制。
