Jokela Maarit, Raki Mari, Heikkinen Kaisa, Sepponen Katri, Eskelinen Anitta, Syväoja Juhani E
Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014, Oulu, Finland.
Protein Expr Purif. 2005 Sep;43(1):73-84. doi: 10.1016/j.pep.2005.05.002.
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.
复制性DNA聚合酶的B亚基属于类钙调神经磷酸酶磷酸二酯酶超家族，从古菌到人类都保守存在。最近我们和其他人表明，古菌D族DNA聚合酶的B亚基（DP1）负责校对3'-5'核酸外切酶活性。B亚基序列的相似性意味着有一个共同的折叠结构，但由于磷酸二酯酶结构域的关键催化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确定。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种不同的重组B亚基。我们发现，可以根据计算出的亲水性平均值（GRAVY）分数预测蛋白质的溶解度。这些分数对于选择能够成功表达的蛋白质很有用。我们优化了詹氏甲烷球菌（Methanococcus jannaschii）DNA聚合酶D的DP1（MjaDP1）的表达和纯化，并表明该蛋白质与一种热稳定的核酸酶活性共同纯化。蛋白质截短实验表明，催化作用不需要N端（第1至134位氨基酸）。该蛋白质含有类钙调神经磷酸酶磷酸二酯酶结构域和OB折叠的C端部分足以产生核酸酶活性。
