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

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

{"title":"根内生真菌 Piriformospora indica 的高亲和性磷酸盐转运体（PiPT）的生化特征。","authors":"","doi":"10.1016/j.pep.2024.106559","DOIUrl":null,"url":null,"abstract":"<div><p>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<sub>max</sub> was measured to be 232.2 ± 11 pmol/min/mg protein. We have found K<sub>m</sub> to be 45.8 ± 6.2 μM suggesting high affinity towards phosphate.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4000,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Biochemical characterization of a high affinity phosphate transporter (PiPT) from root endophyte fungus Piriformospora indica\",\"authors\":\"\",\"doi\":\"10.1016/j.pep.2024.106559\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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<sub>max</sub> was measured to be 232.2 ± 11 pmol/min/mg protein. We have found K<sub>m</sub> to be 45.8 ± 6.2 μM suggesting high affinity towards phosphate.</p></div>\",\"PeriodicalId\":20757,\"journal\":{\"name\":\"Protein expression and purification\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.4000,\"publicationDate\":\"2024-07-31\",\"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/S1046592824001311\",\"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}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein expression and purification","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1046592824001311","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

摘要

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。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

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

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.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

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.