Graduate School of Life Science and Technology, Iryo Sosei University, Iwaki, Fukushima, Japan.
Graduate School of Science and Engineering, Iryo Sosei University, Iwaki, Fukushima, Japan.
Acta Crystallogr F Struct Biol Commun. 2024 Nov 1;80(Pt 11):294-301. doi: 10.1107/S2053230X24009518. Epub 2024 Oct 9.
Proliferating cell nuclear antigen (PCNA) plays a critical role in DNA replication by enhancing the activity of various proteins involved in replication. In this study, the crystal structure of ApePCNA1, one of three PCNAs from the thermophilic archaeon Aeropyrum pernix, was elucidated. ApePCNA1 was cloned and expressed in Escherichia coli and the protein was purified and crystallized. The resulting crystal structure determined at 2.00 Å resolution revealed that ApePCNA1 does not form a trimeric ring, unlike PCNAs from other domains of life. It has unique structural features, including a long interdomain-connecting loop and a PIP-box-like sequence at the N-terminus, indicating potential interactions with other proteins. These findings provide insights into the functional mechanisms of PCNAs in archaea and their evolutionary conservation across different domains of life. A modified medium and protocol were used to express recombinant protein containing the lac operon. The expression of the target protein increased and the total incubation time decreased when using this system compared with those of previous expression protocols.
增殖细胞核抗原（PCNA）在 DNA 复制中发挥关键作用，可增强参与复制的各种蛋白的活性。本研究阐明了嗜热古菌 Aeropyrum pernix 中三种 PCNA 之一 ApePCNA1 的晶体结构。ApePCNA1 在大肠杆菌中进行克隆和表达，然后对蛋白进行纯化和结晶。通过解析 2.00 Å分辨率的晶体结构，发现 ApePCNA1 不像来自其他生命领域的 PCNA 那样形成三聚体环。它具有独特的结构特征，包括长的结构域间连接环和 N 端的 PIP 盒样序列，表明与其他蛋白存在潜在相互作用。这些发现为了解 PCNA 在古菌中的功能机制及其在不同生命领域的进化保守性提供了线索。本研究使用改良的培养基和方案来表达含有 lac 操纵子的重组蛋白。与之前的表达方案相比，使用该系统可增加目标蛋白的表达量并缩短总孵育时间。
