Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped...

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.