Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-04-23 DOI:10.1016/j.pep.2024.106487

Kenji Okumura , Bunzo Mikami , Sayoko Oiki , Kohei Ogura , Wataru Hashimoto

引用次数: 0

Abstract

The bacterial Efe system functions as an importer of free Fe²⁺ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

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用于铁输入的细菌跨膜蛋白 EfeU 的表达、纯化和初步结晶分析。

细菌 Efe 系统的功能是将游离的 Fe2+ 输入细胞，而不依赖于铁螯合化合物（如苷元），它由铁结合蛋白 EfeO、过氧化物酶 EfeB 和跨膜渗透酶 EfeU 组成。我们和其他研究人员报告了 EfeO 和 EfeB 的晶体结构，但 EfeU 的晶体结构仍未确定。在本研究中，我们构建了来源于大肠杆菌的 EfeU 表达系统，选择大肠杆菌 Rosetta-gami 2 (DE3) 作为表达宿主，并成功地纯化了蛋白，通过蓝色原生 PAGE 显示其形成了寡聚体。我们获得了 X 射线晶体学的初步数据，这表明我们在本研究中建立的表达和纯化方法能够对细菌 Efe 系统进行结构分析。

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and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.