Cell-Free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs.

Proton coupled folate transporter (PCFT) is an integral membrane protein with 12 transmembrane segments localized to the plasma membrane. PCFT is the main route by which folate, vitamin B9, from dietary sources enters mammalian cells in the small intestine. Loss-of-function mutations in this membrane transport protein cause hereditary folate malabsorption, and upregulation of PCFT has been reported in cancer cells. Currently, a complete translocation mechanism of folate via PCFT is still missing. To reveal this mechanism via studies of structural architecture and structure-function relationships, soluble and stable PCFT in a phospholipid bilayer environment is needed. We therefore develop an approach to screen lipid environments in which PCFT is most soluble. Traditional in vitro expression and reconstitution into lipid bilayers of integral membrane proteins requires separate steps, which are costly and time-consuming. In this chapter, we describe a protocol for in vitro translation of PCFT into preformed lipid nanodiscs using a cell-free expression system, which helps to accelerate and reduce the cost of the sample preparation.