{"title":"Counterintuitive method improves yields of isotopically labelled proteins expressed in flask-cultured Escherichia coli.","authors":"Miguel Ángel Treviño","doi":"10.1007/s10858-025-00461-2","DOIUrl":null,"url":null,"abstract":"<p><p>NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labelling with <sup>15</sup>N and <sup>13</sup>C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using <sup>13</sup>C-D-glucose and <sup>15</sup>NH<sub>4</sub>Cl as the sole nutrient sources. In recent years, the price of <sup>13</sup>C-D-glucose has almost tripled, making it essential to develop labelling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of <sup>13</sup>C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low <sup>13</sup>C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":" ","pages":""},"PeriodicalIF":1.3000,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Biomolecular NMR","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s10858-025-00461-2","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labelling with 15N and 13C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using 13C-D-glucose and 15NH4Cl as the sole nutrient sources. In recent years, the price of 13C-D-glucose has almost tripled, making it essential to develop labelling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of 13C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low 13C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.
期刊介绍：
The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include:
Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR.
New NMR techniques for studies of biological macromolecules.
Novel approaches to computer-aided automated analysis of multidimensional NMR spectra.
Computational methods for the structural interpretation of NMR data, including structure refinement.
Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals.
New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
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