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bioRxiv · 10.1101/456087

Cell-free soluble expression of the membrane protein PsbS

Krishnan, M.·De Leeuw, F. T.·Pandit, A.
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Abstract

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/{micro}l using a continuous-exchange method at 30{degrees}C, along with a successful purification and refolding of PsbS in n-Dodecyl {beta}-D-maltoside ({beta}-DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.

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Krishnan, M., De Leeuw, F. T., Pandit, A.. 2018-10-29. Cell-free soluble expression of the membrane protein PsbS. https://doi.org/10.1101/456087

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