{"title":"用于胆红素纳米级分析的重组融合蛋白 HUG 的标准化实验室规模生产","authors":"Paola Sist, Suman Saeed, Federica Tramer, Antonella Bandiera, Sabina Passamonti","doi":"10.1016/j.mex.2024.103001","DOIUrl":null,"url":null,"abstract":"<div><div>The recombinant bifunctional protein HELP-UnaG (HUG) is a fusion product of the Human Elastin-like Polypeptide (HELP) with the bilirubin-binding fluorescent protein UnaG. HUG is used for the fluorometric detection of bilirubin in serum and a variety of biological fluids and extracts. Here we describe a detailed method for the standardized production and purification of HUG from <em>E. coli</em> extracts on a laboratory scale. This method takes advantage of the HELP-specific thermoreactive behavior that enables the separation of HUG from complex <em>E. coli</em> extracts by repeated precipitation/re-dissolution steps at near physiological temperature.<ul><li><span>•</span><span><div>The method is based on the inverse thermal transition process.</div></span></li><li><span>•</span><span><div>The “green” method is affordable for basic laboratories and can be easily transferred to new users.</div></span></li></ul></div></div>","PeriodicalId":18446,"journal":{"name":"MethodsX","volume":"13 ","pages":"Article 103001"},"PeriodicalIF":1.6000,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Standardized lab-scale production of the recombinant fusion protein HUG for the nanoscale analysis of bilirubin\",\"authors\":\"Paola Sist, Suman Saeed, Federica Tramer, Antonella Bandiera, Sabina Passamonti\",\"doi\":\"10.1016/j.mex.2024.103001\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>The recombinant bifunctional protein HELP-UnaG (HUG) is a fusion product of the Human Elastin-like Polypeptide (HELP) with the bilirubin-binding fluorescent protein UnaG. HUG is used for the fluorometric detection of bilirubin in serum and a variety of biological fluids and extracts. Here we describe a detailed method for the standardized production and purification of HUG from <em>E. coli</em> extracts on a laboratory scale. This method takes advantage of the HELP-specific thermoreactive behavior that enables the separation of HUG from complex <em>E. coli</em> extracts by repeated precipitation/re-dissolution steps at near physiological temperature.<ul><li><span>•</span><span><div>The method is based on the inverse thermal transition process.</div></span></li><li><span>•</span><span><div>The “green” method is affordable for basic laboratories and can be easily transferred to new users.</div></span></li></ul></div></div>\",\"PeriodicalId\":18446,\"journal\":{\"name\":\"MethodsX\",\"volume\":\"13 \",\"pages\":\"Article 103001\"},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2024-10-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"MethodsX\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2215016124004527\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"MethodsX","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2215016124004527","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
Standardized lab-scale production of the recombinant fusion protein HUG for the nanoscale analysis of bilirubin
The recombinant bifunctional protein HELP-UnaG (HUG) is a fusion product of the Human Elastin-like Polypeptide (HELP) with the bilirubin-binding fluorescent protein UnaG. HUG is used for the fluorometric detection of bilirubin in serum and a variety of biological fluids and extracts. Here we describe a detailed method for the standardized production and purification of HUG from E. coli extracts on a laboratory scale. This method takes advantage of the HELP-specific thermoreactive behavior that enables the separation of HUG from complex E. coli extracts by repeated precipitation/re-dissolution steps at near physiological temperature.
•
The method is based on the inverse thermal transition process.
•
The “green” method is affordable for basic laboratories and can be easily transferred to new users.
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