Detergent-free functionalisation of hybrid vesicles with membrane proteins using SMALPs

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis and synthetic biology. However, functionalisation of HVs remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid lipid particles (SMALPs), we show that a membrane protein (cytochrome bo3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% and without the requirement of any detergent, long incubation times or mechanical disruption. Interestingly, direct transfer of membrane proteins using this approach was not possible into liposomes. This suggests that the HVs are more amenable than liposomes to membrane protein incorporation from a SMALP system. Finally, we show that this transfer method is not limited to cytochrome bo3 and can also be performed with complex membrane protein mixtures.