Penheiter A R, Klucas R V, Sarath G
Department of Biochemistry, University of Nebraska-Lincoln, Nebraska, Lincoln, 68588-0664, USA.
Protein Expr Purif. 1998 Oct;14(1):125-30. doi: 10.1006/prep.1998.0935.
Soybean root nodules possess a developmentally regulated acid phosphatase (ACP) that exhibits the highest specificity for purine 5'-nucleoside monophosphates. The enzyme is a glycosylated dimer of 28- and 31-kDa subunits, which appear to be products of the same gene but differ in posttranslational modifications. In order to perform directed mutagenesis and more extensive biochemical characterization, a means of producing recombinant ACP was needed. Several attempts were made to express ACP in Escherichia coli, but all conditions employed resulted in protein that was found entirely in inclusion bodies, and resolubilization experiments were unsuccessful. Therefore, the methyltrophic yeast Pichia pastoris was chosen as a eukaryotic expression host. The coding sequence of ACP was cloned into the pPIC9 vector to create a fusion with the yeast alpha mating factor secretion signal. The ACP:pPIC9 construct was integrated into P. pastoris strain GS115. Expression of ACP was under the control of an alcohol oxidase methanol-inducible promoter. Methanol induction resulted in secretion of ACP to a level of 10 mg/L. The recombinant ACP was purified 550-fold to homogeneity by phenyl-Sepharose, hydroxyapatite, and MonoS chromatography. The purified enzyme had Km values of 0.08 and 0.12 for 5'-AMP and 5'-GMP. These values were similar to those obtained for the native ACP heterodimer purified from soybean (0.08 and 0.15 mM for 5'-AMP and 5'-GMP). The specific activity of the recombinant enzyme for all substrates tested was 1.6- to 1.8-fold higher than the values for the purified soybean heterodimer.
大豆根瘤含有一种受发育调控的酸性磷酸酶（ACP），该酶对嘌呤5'-核苷单磷酸表现出最高的特异性。该酶是由28 kDa和31 kDa亚基组成的糖基化二聚体，这两个亚基似乎是同一基因的产物，但在翻译后修饰上有所不同。为了进行定向诱变和更广泛的生化特性分析，需要一种生产重组ACP的方法。人们尝试了多种在大肠杆菌中表达ACP的方法，但所有采用的条件都导致蛋白质完全存在于包涵体中，而且复性实验也未成功。因此，选择甲基营养型酵母毕赤酵母作为真核表达宿主。将ACP的编码序列克隆到pPIC9载体中，与酵母α交配因子分泌信号形成融合。ACP:pPIC9构建体整合到毕赤酵母菌株GS115中。ACP的表达受甲醇诱导型醇氧化酶启动子的控制。甲醇诱导导致ACP分泌到10 mg/L的水平。通过苯基琼脂糖凝胶、羟基磷灰石和MonoS色谱将重组ACP纯化了550倍，达到同质。纯化后的酶对5'-AMP和5'-GMP的Km值分别为0.08和0.12。这些值与从大豆中纯化的天然ACP异二聚体的值相似（5'-AMP和5'-GMP分别为0.08和0.15 mM）。重组酶对所有测试底物的比活性比纯化后的大豆异二聚体的值高1.6至1.8倍。
