Nanodiscs technology provides a platform for studying membrane proteins in a controlled, native-like lipid environment. However, the membrane scaffold proteins (MSPs) essential for nanodiscs assembly are often prone to proteolysis and polymerization, which can hinder nanodiscs formation. Additionally, traditional membrane protein reconstitution methods are time-consuming and produce low yields, limiting the application of many biophysical techniques that require large amounts of material. In this study, we present an optimized protocol for the expression and purification of MSPs that significantly reduces proteolytic degradation and polymerization. Additionally, we introduce a rapid and efficient reconstitution method using PD-10 desalting columns, to produce large quantities of nanodiscs assembly. This improved workflow enhances both the yield and quality of nanodiscs with the membrane protein successfully inserted. Using this protocol, we successfully reconstituted two membrane proteins, KcsA and ABCB10, and confirmed their function in our nanodiscs. Our approach provides a scalable and reliable platform for membrane protein research, expanding access to biophysical analyses that were previously limited by sample constraints.
Skains et al. (2026) studied this question.