Chassin Yael, Kapri-Pardes Einat, Sinvany Galit, Arad Tal, Adam Zach
Institute of Plant Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Plant Physiol. 2002 Oct;130(2):857-64. doi: 10.1104/pp.007922.
The Arabidopsis genome contains 14 genes encoding the serine protease DegP. Products of four of these genes are located in the chloroplast: three in the thylakoid lumen and one on the stromal side of the membrane. We expressed the gene encoding DegP1 as a His-tagged fusion protein in Escherichia coli, purified the protein by affinity chromatography, and characterized it biochemically. Size-exclusion chromatography suggested that DegP1 eluted from the column as a mixture of monomers and hexamers. Proteolytic activity was characterized using beta-casein as a model substrate. DegP1 demonstrated concentration-dependent activity, a pH optimum of 6.0 and increasing activity at elevated temperatures. DegP1 was capable of degrading two lumenal proteins, plastocyanin and OE33, suggesting a role as a general-purpose protease in the thylakoid lumen. The results of this work are discussed in the context of the recent elucidation of the structure of the E. coli homolog and the possible physiological role of the protease in the chloroplast lumen.
拟南芥基因组包含14个编码丝氨酸蛋白酶DegP的基因。其中四个基因的产物位于叶绿体中：三个位于类囊体腔，一个位于膜的基质侧。我们在大肠杆菌中表达了编码DegP1的基因，将其作为带有His标签的融合蛋白，通过亲和层析纯化该蛋白，并对其进行生化特性分析。尺寸排阻色谱表明，DegP1从柱中洗脱时为单体和六聚体的混合物。使用β-酪蛋白作为模型底物对蛋白水解活性进行了表征。DegP1表现出浓度依赖性活性，最适pH为6.0，在升高温度时活性增加。DegP1能够降解两种腔蛋白，质体蓝素和OE33，表明其在类囊体腔中作为通用蛋白酶发挥作用。本文的研究结果结合最近对大肠杆菌同源物结构的阐明以及该蛋白酶在叶绿体腔中可能的生理作用进行了讨论。
