Layara Akemi Abiko, Marco Rogowski, Antoine Gautier, Gebhard Schertler, Stephan Grzesiek
{"title":"用于结构研究的功能性G蛋白偶联受体在大肠杆菌中的高效生产","authors":"Layara Akemi Abiko, Marco Rogowski, Antoine Gautier, Gebhard Schertler, Stephan Grzesiek","doi":"10.1007/s10858-020-00354-6","DOIUrl":null,"url":null,"abstract":"<p>G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in <i>E. coli</i> has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR <i>E. coli</i> expression and then describe the development of an optimized robust protocol for the <i>E. coli</i> expression and purification of two mutants of the turkey β<sub>1</sub>-adrenergic receptor (β<sub>1</sub>AR) uniformly or selectively labeled in <sup>15</sup>N or <sup>2</sup>H,<sup>15</sup>N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for <i>E. coli</i> expression. Optimization of <i>E. coli</i> expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2–0.3?mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β<sub>1</sub>AR mutant also comprises the two native tyrosines Y<sup>5.58</sup> and Y<sup>7.53</sup>, which enable G protein binding. High-quality <sup>1</sup>H-<sup>15</sup>N TROSY spectra were obtained for <i>E. coli</i>-expressed YY-β<sub>1</sub>AR in three different functional states (antagonist, agonist, and agonist?+?G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.</p>","PeriodicalId":613,"journal":{"name":"Journal of Biomolecular NMR","volume":"75 1","pages":"25 - 38"},"PeriodicalIF":1.3000,"publicationDate":"2021-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/s10858-020-00354-6","citationCount":"7","resultStr":"{\"title\":\"Efficient production of a functional G protein-coupled receptor in E. coli for structural studies\",\"authors\":\"Layara Akemi Abiko, Marco Rogowski, Antoine Gautier, Gebhard Schertler, Stephan Grzesiek\",\"doi\":\"10.1007/s10858-020-00354-6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in <i>E. coli</i> has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR <i>E. coli</i> expression and then describe the development of an optimized robust protocol for the <i>E. coli</i> expression and purification of two mutants of the turkey β<sub>1</sub>-adrenergic receptor (β<sub>1</sub>AR) uniformly or selectively labeled in <sup>15</sup>N or <sup>2</sup>H,<sup>15</sup>N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for <i>E. coli</i> expression. Optimization of <i>E. coli</i> expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2–0.3?mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β<sub>1</sub>AR mutant also comprises the two native tyrosines Y<sup>5.58</sup> and Y<sup>7.53</sup>, which enable G protein binding. High-quality <sup>1</sup>H-<sup>15</sup>N TROSY spectra were obtained for <i>E. coli</i>-expressed YY-β<sub>1</sub>AR in three different functional states (antagonist, agonist, and agonist?+?G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.</p>\",\"PeriodicalId\":613,\"journal\":{\"name\":\"Journal of Biomolecular NMR\",\"volume\":\"75 1\",\"pages\":\"25 - 38\"},\"PeriodicalIF\":1.3000,\"publicationDate\":\"2021-01-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1007/s10858-020-00354-6\",\"citationCount\":\"7\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Biomolecular NMR\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s10858-020-00354-6\",\"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}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Biomolecular NMR","FirstCategoryId":"99","ListUrlMain":"https://link.springer.com/article/10.1007/s10858-020-00354-6","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}
Efficient production of a functional G protein-coupled receptor in E. coli for structural studies
G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey β1-adrenergic receptor (β1AR) uniformly or selectively labeled in 15N or 2H,15N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2–0.3?mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β1AR mutant also comprises the two native tyrosines Y5.58 and Y7.53, which enable G protein binding. High-quality 1H-15N TROSY spectra were obtained for E. coli-expressed YY-β1AR in three different functional states (antagonist, agonist, and agonist?+?G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.
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.