{"title":"Standardized lab-scale production of the recombinant fusion protein HUG for the nanoscale analysis of bilirubin","authors":"","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":null,"pages":null},"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}
引用次数: 0
Abstract
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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