Purification, folding, activity analysis and substrate specificity of Pseudomonas diacylglycerol kinase

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-04-27 DOI:10.1016/j.pep.2025.106723

Yipeng Chen , Bin Huai , Jin Chuan Wu , Ning Zhang , Yong Wang , Qingxin Li

{"title":"Purification, folding, activity analysis and substrate specificity of Pseudomonas diacylglycerol kinase","authors":"Yipeng Chen , Bin Huai , Jin Chuan Wu , Ning Zhang , Yong Wang , Qingxin Li","doi":"10.1016/j.pep.2025.106723","DOIUrl":null,"url":null,"abstract":"<div><div>The structural and functional investigation of bacterial membrane proteins is crucial to the development of antibiotics. Diacylglycerol kinase (DAGK) from <em>Escherichia coli</em> (<em>E. coli</em>) has been extensively studied as a model membrane protein. However, the DAGK from <em>Pseudomonas aeruginosa</em> (PAO1-DAGK) with a 44 % sequence identity to <em>E. coli</em>-DAGK is not well characterized. To explore the properties of PAO1-DAGK, it was successfully expressed in <em>E. coli</em> and was purified in Decyl-β-D-maltoside (DM) micelles followed with characterizations. Chemical cross-linking studies revealed that PAO1-DAGK in DM micelles could form dimers and trimers. The kinase activity of PAO1-DAGK was determined to be 24.2 ± 2.2 U/mg protein in a mixed-micelle system. The effects of pH and temperature on the activity of PAO1-DAGK were also investigated, respectively. PAO1-DAGK in DM micelles exhibited good stability at pH 6.0–10.0 and below 45 °C. Substrate specificity measurements indicated that PAO1-DAGK demonstrated a clear preference for medium-chain diacylglycerols (DAGs) in the mixed-micelle system, with sn-1,2-Dihexanoylglycerol (DiC6) being the most favored substrate. Molecular docking results demonstrated the interactions between DAGs and PAO1-DAGK.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"232 ","pages":"Article 106723"},"PeriodicalIF":1.4000,"publicationDate":"2025-04-27","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/S1046592825000658","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 structural and functional investigation of bacterial membrane proteins is crucial to the development of antibiotics. Diacylglycerol kinase (DAGK) from Escherichia coli (E. coli) has been extensively studied as a model membrane protein. However, the DAGK from Pseudomonas aeruginosa (PAO1-DAGK) with a 44 % sequence identity to E. coli-DAGK is not well characterized. To explore the properties of PAO1-DAGK, it was successfully expressed in E. coli and was purified in Decyl-β-D-maltoside (DM) micelles followed with characterizations. Chemical cross-linking studies revealed that PAO1-DAGK in DM micelles could form dimers and trimers. The kinase activity of PAO1-DAGK was determined to be 24.2 ± 2.2 U/mg protein in a mixed-micelle system. The effects of pH and temperature on the activity of PAO1-DAGK were also investigated, respectively. PAO1-DAGK in DM micelles exhibited good stability at pH 6.0–10.0 and below 45 °C. Substrate specificity measurements indicated that PAO1-DAGK demonstrated a clear preference for medium-chain diacylglycerols (DAGs) in the mixed-micelle system, with sn-1,2-Dihexanoylglycerol (DiC6) being the most favored substrate. Molecular docking results demonstrated the interactions between DAGs and PAO1-DAGK.

查看原文本刊更多论文

假单胞菌二酰基甘油激酶的纯化、折叠、活性分析和底物特异性

细菌膜蛋白的结构和功能研究对抗生素的开发至关重要。大肠杆菌二酰基甘油激酶（DAGK）作为一种模式膜蛋白已被广泛研究。然而，铜绿假单胞菌的DAGK （PAO1-DAGK）与大肠杆菌的DAGK序列同源性为44%，尚未得到很好的鉴定。为了探究PAO1-DAGK的性质，我们成功地在大肠杆菌中表达了PAO1-DAGK，并用Decyl-β- d -麦芽糖苷（DM）胶束纯化了PAO1-DAGK，并对其进行了表征。化学交联研究表明，PAO1-DAGK在DM胶束中可以形成二聚体和三聚体。在混合胶束体系中测定PAO1-DAGK的激酶活性为24.2±2.2 U/mg蛋白。研究了pH和温度对PAO1-DAGK活性的影响。DM胶束中的PAO1-DAGK在pH 6.0 ~ 10.0和45℃以下具有良好的稳定性。底物特异性测量表明，PAO1-DAGK在混合胶束体系中对中链二酰基甘油（dag）表现出明显的偏好，其中sn-1,2-二己醇甘油（DiC6）是最受青睐的底物。分子对接结果表明，dag与PAO1-DAGK之间存在相互作用。

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分钟内获得全文求助全文

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.