| Chapter title |
High-Throughput Production and Optimization of Membrane Proteins After Expression in Mammalian Cells.
|
|---|---|
| Chapter number | 5 |
| Book title |
Advanced Methods in Structural Biology
|
| Published in |
Methods in molecular biology, January 2023
|
| DOI | 10.1007/978-1-0716-3147-8_5 |
| Pubmed ID | |
| Book ISBNs |
978-1-07-163146-1, 978-1-07-163147-8
|
| Authors |
Gamage, Nadisha, Cheruvara, Harish, Harrison, Peter J, Birch, James, Hitchman, Charlie J, Olejnik, Monika, Owens, Raymond J, Quigley, Andrew, Harrison, Peter J., Hitchman, Charlie J., Owens, Raymond J. |
| Abstract |
High-quality protein samples are an essential requirement of any structural biology experiment. However, producing high-quality protein samples, especially for membrane proteins, is iterative and time-consuming. Membrane protein structural biology remains challenging due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Overcoming the twin problems of compositional and conformational instability requires an understanding of protein size, thermostability, and sample heterogeneity, while a parallelized approach enables multiple conditions to be analyzed simultaneously. We present a method that couples the high-throughput cloning of membrane protein constructs with the transient expression of membrane proteins in human embryonic kidney (HEK) cells and rapid identification of the most suitable conditions for subsequent structural biology applications. This rapid screening method is used routinely in the Membrane Protein Laboratory at Diamond Light Source to identify the most successful protein constructs and conditions while excluding those that will not work. The 96-well format is easily adaptable to enable the screening of constructs, pH, salts, encapsulation agents, and other additives such as lipids. |
X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United States | 1 | 50% |
| Unknown | 1 | 50% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Scientists | 1 | 50% |
| Members of the public | 1 | 50% |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 4 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Master | 1 | 25% |
| Unknown | 3 | 75% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 1 | 25% |
| Unknown | 3 | 75% |