中央研究院 原子與分子科學研究所

Lipid nanodiscs are widely used platforms for studying membrane proteins in a near-native environment. Lipid nanodiscs made with membrane scaffold proteins (MSPs) in linear form have been well studied. Recently, a new kind of nanodisc made with MSPs in circular form, referred to as covalently circularized nanodiscs (cND), and is reported to have some possible advantages in various applications. Given the potential of nanodisc technology, researchers in the field are very interested in learning more about this new kind of nanodisc, such as the reproducibility, the production yield, and the possible pros and cons of using it. However, research on these issues is lacking. Here we report a new study on nanodiscs made with circular MSPs, which are produced from a method different to the previously reported method. We show that our novel production method, detergent-assisted sortase-mediated ligation, can effectively avoid high-molecular weight byproducts, and also significantly improves the yield of the target proteins up to around eighty percent for larger circular MSP constructs. In terms of the application of circular MSPs, we demonstrate that they can be used to assemble nanodiscs using both synthetic lipids and native lipid extract as the source of lipids. We also show that bacteriorhodopsin (bR) can be successfully incorporated into this new kind of cND. Moreover, we found that cNDs have improved stability against both heat and high-concentration induced aggregations, making them more beneficial for related applications.

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