Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis
Expression and characterization of the thylakoid lumen protease DegP1 from Arabidopsis
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DOI：
10.1104/pp.007922
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发表时间：
2002-10-01
期刊：
影响因子：
7.4
通讯作者：
Adam, Z
中科院分区：
文献类型：
--
作者：
Chassin, Y;Kapri-Pardes, E;Adam, Z
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：三个位于类囊体腔中，一个位于膜的基质侧。我们在大肠杆菌中将编码 DegP1 的基因表达为 His 标记的融合蛋白，通过亲和层析纯化该蛋白，并对其进行生化表征。尺寸排阻色谱表明 DegP1 作为单体和六聚体的混合物从柱中洗脱。使用β-酪蛋白作为模型底物来表征蛋白水解活性。 DegP1 表现出浓度依赖性活性，最适 pH 值为 6.0，并且在升高的温度下活性增加。 DegP1 能够降解两种腔蛋白：质体蓝蛋白和 OE33，表明其在类囊体腔中具有通用蛋白酶的作用。这项工作的结果是在最近阐明大肠杆菌同源物的结构以及蛋白酶在叶绿体腔中可能的生理作用的背景下讨论的。
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.