The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit
The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit
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DOI：
10.1016/j.pep.2005.05.002
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发表时间：
2005-09-01
影响因子：
1.6
通讯作者：
Syväoja, JE
中科院分区：
文献类型：
--
作者：
Jokela, M;Raki, M;Syväoja, JE
复制型DNA聚合酶的B亚基属于钙调神经磷酸酶超家族，从古生物到人类都是保守的。最近，我们和其他人发现，古生菌家族D DNA聚合酶的B亚单位(DP1)负责校对3‘-5’外切酶的活性。B-亚基序列的相似性意味着有一个共同的折叠，但由于磷酸酯酶结构域的关键催化残基和金属结合残基在真核生物的B-亚基中被破坏，它们的共同功能尚未被鉴定。为了更详细地研究B亚基的结构和活性，我们在大肠杆菌中表达了13个不同的重组B亚基。我们发现，蛋白质的溶解度可以从计算的肉汁分数中预测出来。这些分数对选择成功表达的蛋白质是有用的。我们对DNA聚合酶D(MjaDP1)的表达和纯化进行了优化，结果表明该蛋白具有较高的热稳定性核酸酶活性。蛋白质的截短表明N末端(aa1-134)不是催化所必需的。含有钙调神经磷酸酶结构域和OB-折叠的蛋白质的C-末端部分足以满足核酸酶的活性。(C)2005 Elsevier Inc.保留所有权利。
The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The similarity of B-subunit sequences implies a common fold, but since the key catalytic and metal binding residues of the phosphoesterase domain are disrupted in the eukaryotic B-subunits, their common function has not been identified. To study the structure and activities of B-subunits in more detail, we expressed 13 different recombinant B-subunits in Escherichia coli. We found that the solubility of a protein could be predicted from the calculated GRAVY score. These scores were useful for the selection of proteins for successful expression. We optimized the expression and purification of Methanocaldococcus (Methanococcus)jannaschii DP1 of DNA polymerase D (MjaDP1) and show that the protein co-purifies with a thermostable nuclease activity. Truncation of the protein indicates that the N-terminus (aa 1- 134) is not needed for catalysis. The C-terminal part of the protein containing both the calcineurin-like phosphoesterase domain and the OB-fold is sufficient for the nuclease activity. (c) 2005 Elsevier Inc. All rights reserved.