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

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

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引用次数: 0

摘要

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。

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

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.

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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.