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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.

Original publication

DOI

10.1007/978-1-0716-3147-8_5

Type

Journal article

Journal

Methods in molecular biology (Clifton, N.J.)

Publication Date

01/2023

Volume

2652

Pages

79 - 118

Addresses

Membrane Protein Laboratory, Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, UK.

Keywords

Animals, Mammals, Humans, Membrane Proteins