Cell-free soluble expression of the membrane protein PsbS.
In-vitro refolding
Light harvesting
Photosystem II subunit S
pH sensor
Journal
Protein expression and purification
ISSN: 1096-0279
Titre abrégé: Protein Expr Purif
Pays: United States
ID NLM: 9101496
Informations de publication
Date de publication:
07 2019
07 2019
Historique:
received:
14
11
2018
revised:
13
02
2019
accepted:
13
02
2019
pubmed:
23
2
2019
medline:
28
4
2020
entrez:
22
2
2019
Statut:
ppublish
Résumé
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. Here we present cell-free (CF) expression as a successful method for in vitro production of PsbS that would allow such incorporation. The addition of several detergents, liposomes and lipid nanodiscs was tested for achieving soluble CF expression of PsbS. We have optimized the CF 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.
Identifiants
pubmed: 30790615
pii: S1046-5928(18)30542-4
doi: 10.1016/j.pep.2019.02.010
pii:
doi:
Substances chimiques
Glucosides
0
Lipids
0
Liposomes
0
Photosystem II Protein Complex
0
dodecyl maltoside
69227-93-6
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
17-20Informations de copyright
Copyright © 2019. Published by Elsevier Inc.