Expression and purification of the intact bacterial ergothioneine transporter EgtU

IF 1.4 4区 生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-11-20 DOI:10.1016/j.pep.2024.106633
Katherine A. Edmonds, Karla Diaz-Rodriguez, David P. Giedroc
{"title":"Expression and purification of the intact bacterial ergothioneine transporter EgtU","authors":"Katherine A. Edmonds,&nbsp;Karla Diaz-Rodriguez,&nbsp;David P. Giedroc","doi":"10.1016/j.pep.2024.106633","DOIUrl":null,"url":null,"abstract":"<div><div>The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in <em>Streptococcus pneumoniae</em>, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed <em>S. pneumoniae</em> EgtU and purified it from <em>E. coli</em> BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106633"},"PeriodicalIF":1.4000,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824002055","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in Streptococcus pneumoniae, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed S. pneumoniae EgtU and purified it from E. coli BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization.
查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
完整细菌麦角硫因转运体 EgtU 的表达和纯化。
细菌 ATP 结合盒(ABC)转运体 EgtU 负责摄取肺炎链球菌细胞中的抗氧化剂麦角硫因,它在多种微生物病原体中都有同源物,其种类之多令人惊讶。EgtU 的溶质结合结构域的晶体结构已有报道,但完整的异构四聚体转运体的结构和功能的许多细节仍有待阐明。在本研究中,我们表达了肺炎双球菌的 EgtU,并从大肠杆菌 BL21 (DE3) 中纯化出了高纯度和高均匀度的 EgtU。我们的初步数据证实了在 DDM 胶束中溶解的全长转运体与麦角硫因的结合和 ATP 的水解。我们的工作流程可以分离出适当数量的 EgtU,用于正在进行的结构研究和详细的生物物理表征。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
Protein expression and purification
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.
期刊最新文献
Expression and purification of the intact bacterial ergothioneine transporter EgtU Editorial Board Recombinant human FOXJ1 protein binds DNA, forms higher-order oligomers, has gel-shifting domains and contains intrinsically disordered regions Thermostable phenylacetic acid degradation protein TtPaaI from Thermus thermophilus as a scaffold for tetravalent display of proteins The heterogeneous expression, extraction, and purification of recombinant Caldanaerobacter subterraneus subsp. tengcongensis apurine/apyrimidine endonuclease in Escherichia coli
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1