Biochemical characterization of a high affinity phosphate transporter (PiPT) from root endophyte fungus Piriformospora indica

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-07-31 DOI:10.1016/j.pep.2024.106559

Hemant Kumar , Aayushi Bajaj , Paras Kumar , Rupesh Aggarwal , Vinayak Chalia , Rajesh Kumar Pradhan , Ritu Yadav , Shalini Sinha , Vishad Agarwal , William Harries , Meenakshi Dua , Robert M. Stroud , Atul Kumar Johri

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Abstract

We have functionally characterized the high-affinity phosphate transporter (PiPT) from the root endophyte fungus Piriformospora indica. PiPT belongs to the major facilitator superfamily (MFS). PiPT protein was purified by affinity chromatography (Ni-NTA) and Size Exclusion Chromatography (SEC). The functionality of solubilized PiPT was determined in detergent-solubilized state by fluorescence quenching and in proteoliposomes. In the fluorescence quenching assay, PiPT exhibited a saturation concentration of approximately 2 μM, at a pH of 4.5. Proteoliposomes of size 121.6 nm radius, showed transportation of radioactive phosphate. V_max was measured to be 232.2 ± 11 pmol/min/mg protein. We have found K_m to be 45.8 ± 6.2 μM suggesting high affinity towards phosphate.

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根内生真菌 Piriformospora indica 的高亲和性磷酸盐转运体（PiPT）的生化特征。

我们从功能上鉴定了根内生真菌 Piriformospora indica 的高亲和性磷酸盐转运体（PiPT）。PiPT 属于主要促进剂超家族（MFS）。通过亲和层析（Ni-NTA）和尺寸排阻层析（SEC）纯化了 PiPT 蛋白。通过荧光淬灭法测定了在去垢剂溶解状态下和在蛋白脂质体中溶解的 PiPT 的功能。在荧光淬灭试验中，PiPT 在 pH 值为 4.5 时的饱和浓度约为 2 μM。半径为 121.6 纳米的原脂质体显示出放射性磷酸盐的运输。Vmax 测量值为 232.2±11 pmol/min/mg。我们发现 Km 为 45.8±6.2 μM，这表明它对磷酸盐具有很高的亲和力。

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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.