Patterson W R, Poulos T L
Dept. of Physiology and Biophysics, University of California, Irvine 92717.
J Biol Chem. 1994 Jun 24;269(25):17020-4.
An Escherichia coli expression system has been developed for pea cytosolic ascorbate peroxidase (APX). The enzyme was expressed as a fusion product with the E. coli maltose-binding protein for rapid, affinity chromatography purification. Recombinant ascorbate peroxidase (rAPX) was purified by tryptic digestion to separate the maltose-binding protein from rAPX followed by three chromatographic steps. The purified rAPX protein demonstrated identical electrophoretic, enzymatic, and spectral properties when compared to native APX isolated from pea shoots. Upon addition of an equal molar amount of H2O2, rAPX exhibits an initial decrease in the Soret maximum, which slowly converts to a stable, red-shifted Soret peak similar to that observed for cytochrome c peroxidase Compound I, indicating that rAPX Compound I consists of an oxyferryl (Fe(4+)-O) center. rAPX has been crystallized in a form suitable for crystal structure determination, and a preliminary set of native data to 2.6 A have been collected.
已经开发出一种用于豌豆胞质抗坏血酸过氧化物酶（APX）的大肠杆菌表达系统。该酶作为与大肠杆菌麦芽糖结合蛋白的融合产物进行表达，以便通过亲和色谱快速纯化。重组抗坏血酸过氧化物酶（rAPX）通过胰蛋白酶消化进行纯化，以将麦芽糖结合蛋白与rAPX分离，随后进行三个色谱步骤。与从豌豆芽中分离出的天然APX相比，纯化后的rAPX蛋白表现出相同的电泳、酶学和光谱特性。加入等摩尔量的H2O2后，rAPX的Soret最大值最初会下降，随后缓慢转变为稳定的、红移的Soret峰，类似于细胞色素c过氧化物酶化合物I所观察到的情况，这表明rAPX化合物I由一个氧铁（Fe(4+)-O）中心组成。rAPX已结晶成适合晶体结构测定的形式，并已收集到分辨率为2.6 Å的初步原生数据。
