Cell-free soluble expression of the membrane protein PsbS
Introduction
The discovery of Photosystem II subunit S (PsbS) has revealed that it has a prominent role in sensing changes in thylakoidal pH. PsbS brings about structural rearrangements in the neighbouring photosynthetic proteins, leading to de-excitation of the antenna chlorophylls (Chls), which is known as the fast qE phase of non-photochemical quenching (NPQ) [1,2]. PsbS is a 22 KDa membrane protein with four transmembrane helices and belongs to the light harvesting complex (LHC) protein superfamily [3]. PsbS can be overexpressed using E. coli and refolded into helical structures using several types of detergents [4,5]. Although this overexpression method is well established, there are challenges of inclusion bodies production, low yield while isotope and selective labelling, losses upon insertion into liposomes or nanodiscs and toxic effects to the host cells upon overexpression. Cell-free (CF) protein expression has emerged as an alternative technique for production of a diverse range of membrane proteins for functional studies [6,7]. In-vitro refolding of various membrane proteins during the CF reaction can be achieved by adding detergents, liposomes or lipid nanodiscs to the reaction mixture [[7], [8], [9]]. However, the yields for membrane proteins produced using the CF technique is still far below the yields achieved for soluble proteins [10]. We show that PsbS from Physcomitrella patens can be synthesized and successfully refolded by using a commercial CF system. Various detergents and lipid nanodiscs were added to the reaction mixtures to achieve soluble PsbS using fed-batch system. Purification and refolding of both pellet and soluble PsbS produced using CF reactions was successfully achieved in the detergent n-Dodecyl β-D-maltoside (β-DM).
Section snippets
Cell-free (CF) expression protocol
The PsbS gene from Physcomitrella patens [4] was inserted in a pExp5 vector for the CF reactions. Expressway™ Cell-Free Expression System (Invitrogen) was used to carry out the CF reactions We used 25 μl reaction mixture (RM) containing the template DNA plasmid, incubated for 2–4 h at 30 °C or 37 °C unless states otherwise, while shaking at 1200 rpm. After 30 min, 25 μl of feeding mixture (FM) was added into the reaction volume. To produce soluble PsbS, CF reactions were carried out in the
CF synthesis of PsbS as pellet
For carrying out the CF reaction, the PsbS plasmid was added to the CF E. coli reaction mix and incubated at 37 °C for 3 h. Using 1330 ng of plasmid in the CF reaction, a yield of 200 ng/μl PsbS could be produced with this standard reaction protocol (Fig. S1). Since PsbS is a membrane protein, without the presence of detergents, the produced protein formed a pellet, P (Fig. 1a) in the reaction. Fig. 1b shows the purification of PsbS by washing with urea buffer (8 M urea, 100 mM Sodium
Conclusions
We demonstrate that with CF synthesis, PsbS can be produced either as aggregate pellet or in a soluble state in the presence of membrane-mimicking additives, in contrast to the E. coli overexpression, where PsbS is always secreted in inclusion bodies. The CF produced PsbS protein could successfully be refolded into detergent micelles, showing ∼50% helical content. The protocols were optimized to yield ∼500 ng/μl PsbS production in a single reaction, which could be up-scaled for structural
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We would like to thank Nora Goosen and Geri Moolenaar for their technical help and advice and Emanuela Crisafi for providing the liposomes. A.P. and M.K. were financially supported by a CW-VIDI grant of the Netherlands Organization of Scientific Research (grant nr. 723.012.103).
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