Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-03-22 DOI:10.1016/j.pep.2024.106476

Yitong Zhang , Yangjie Ren , Ben Wang, Shiyang Guo, Siqi Wang, Jinglin Jin, Lihong Yang, Wei Gao

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引用次数: 0

Abstract

Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn²⁺ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.

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肺炎衣原体 AP 内切酶 IV 的纯化、结晶和初步晶体学分析。

碱基切除是核苷酸切除过程中由内切酶 IV 介导的重要 DNA 修复过程。在肺炎衣原体中，CpendoIV是唯一的AP内切酶IV，具有DNA复制防错功能，因此是抗衣原体药物开发的一个很有前景的靶点。为了预测 CpendoIV 的结构，研究人员与 DNA 进行了分子对接，分析了复杂的结合位点和蛋白质表面静电势。克隆了CpendoIV，在大肠杆菌中表达，并通过Ni-NTA螯合和大小排阻色谱进行纯化。低浓度的 NaCl 会导致纯化过程中的聚集，而高浓度的 NaCl 则会提高纯度。CpendoIV 能识别并裂解 dsDNA 上的 AP 位点，Zn2+ 会影响其活性。在 pH 值为 5.2、25%（w/v）聚乙二醇 3350、8%（v/v）Tacsimate 的条件下，CpendoIV 实现了结晶，并在 100 K 时获得了 1.91 Å 分辨率的 X 射线衍射数据。这项研究的重要意义在于加深了人们对 CpendoIV 参与碱基切除修复过程的了解，为深入研究肺炎衣原体提供了线索。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.