| Chapter title |
High-Efficiency Expression of Yeast-Derived G-Protein Coupled Receptors and 19F Labeling for Dynamical Studies
|
|---|---|
| Chapter number | 19 |
| Book title |
Protein NMR
|
| Published in |
Methods in molecular biology, January 2018
|
| DOI | 10.1007/978-1-4939-7386-6_19 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7385-9, 978-1-4939-7386-6
|
| Authors |
Libin Ye, Alexander P. Orazietti, Aditya Pandey, R. Scott Prosser, Ye, Libin, Orazietti, Alexander P., Pandey, Aditya, Prosser, R. Scott |
| Abstract |
We describe a detailed protocol for heterologous expression of the human adenosine A2A G-protein coupled receptor (GPCR), using Pichia pastoris. Details are also provided for the reconstitution and functional purification steps. Yields of 2-6 mg/g membrane were obtained prior to functional purification (ligand column purification). Typically, functional purification reduced overall yields by a factor of 2-4, resulting in final functional production of 0.5-3 mg/L membrane. Yeast is an excellent protein expression system for NMR given its high tolerance for isotope-enriched solvents and its ability to grow in minimal media. |
X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Japan | 1 | 25% |
| United States | 1 | 25% |
| Unknown | 2 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 4 | 100% |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 8 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 3 | 38% |
| Professor | 1 | 13% |
| Student > Ph. D. Student | 1 | 13% |
| Student > Master | 1 | 13% |
| Unknown | 2 | 25% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 4 | 50% |
| Chemistry | 1 | 13% |
| Unknown | 3 | 38% |