Cell-free soluble expression of the membrane protein PsbS

Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photochemical quenching for photoprotection of plants under high-light conditions. The activation mechanism of PsbS and its pH-induced conformational changes are currently unknown. For structural investigation of PsbS, effective synthesis of PsbS with selective isotope or electron-spin labels or non-natural amino acids incorporated would be a great asset. This communication presents cell-free expression as a successful method for in vitro production of PsbS that would allow such incorporation. We have optimized the cell-free method to yield soluble PsbS of ~500 ng/µl using a continuous-exchange method at 30°C, along with a successful purification and refolding of PsbS in n-Dodecyl β -D-maltoside ( β -DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.