Hermanson I L, Turchi J J
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
Protein Expr Purif. 2000 Jun;19(1):1-11. doi: 10.1006/prep.2000.1224.
The xeroderma pigmentosum group A protein (XPA) is an essential component of the eukaryotic nucleotide excision repair (NER) process. Recombinant human XPA was expressed in baculovirus-infected insect cells as a His-tagged fusion protein. A two-column purification procedure resulted in greater than 90% purity for the recombinant protein with a final yield of 0.53 mg from 200 ml of infected cells. The recombinant protein migrated as a doublet of 44 and 42 kDa upon SDS-PAGE consistent with that observed for the native protein. XPA can interact with a number of proteins including replication protein A (RPA) which has been implicated in the initial recognition of damaged DNA. Using a modified ELISA, we demonstrate that the recombinant XPA fusion protein also forms a complex with RPA independent of DNA. The ability of XPA to bind damaged DNA was assessed in an electrophoretic mobility shift assay using globally cisplatin-damaged DNA. The results revealed a slight preference for DNA damaged with cisplatin consistent with its proposed role in the recognition of damaged DNA. The recombinant XPA fusion protein was able to complement cell-free extracts immunodepleted of XPA restoring NER-catalyzed incision of cisplatin-damaged DNA in an in vitro excision repair assay.
着色性干皮病 A 组蛋白（XPA）是真核生物核苷酸切除修复（NER）过程的重要组成部分。重组人 XPA 在杆状病毒感染的昆虫细胞中表达为一种带有His标签的融合蛋白。通过两步柱纯化程序，重组蛋白的纯度超过 90%，从 200 ml 感染细胞中最终获得 0.53 mg 的产量。在 SDS-PAGE 上，重组蛋白迁移为 44 kDa 和 42 kDa 的双峰，与天然蛋白一致。XPA 可与多种蛋白质相互作用，包括参与受损 DNA 初始识别的复制蛋白 A（RPA）。使用改良的 ELISA，我们证明重组 XPA 融合蛋白也能独立于 DNA 与 RPA 形成复合物。使用经顺铂整体损伤的 DNA 通过电泳迁移率变动分析评估 XPA 结合受损 DNA 的能力。结果显示对顺铂损伤的 DNA 略有偏好，与其在识别受损 DNA 中的假定作用一致。在体外切除修复试验中，重组 XPA 融合蛋白能够补充 XPA 免疫耗尽的无细胞提取物，恢复 NER 催化的顺铂损伤 DNA 的切口。
