Overexpression and purification of recombinant Deg proteases from Synechocystis 6803 The construct overexpressing Synechocystis rHhoA protease and lacking the predicted signal peptide at the N-terminal end was developed by Huesgen et al. [72]. Cloning and purification of recombinant HtrA and HhoB proteases followed a similar strategy [50]. Transformed E. coli cells were grown in LB liquid medium containing 100 mg ml21 ampicillin at 19uC for 10 h, and expression of recombinant proteases was induced by the addition of 0.1 mM isopropyl-1-thio-D-galactoside (IPTG). Cultures were further grown overnight at 19uC, then harvested by centrifugation (5 000 g for 10 min at 4uC). Cell pellets were resuspended in 10 to 20 ml of binding buffer (50 mM HEPES-NaOH, pH 8.0 and 300 mM NaCl) and stored at 220uC. Cells were lyzed on ice by 10 to 15 repeats of 10-s sonication with 20-s cooling interval and centrifuged (26 000 g, 1 h, 4uC). Recombinant proteases were purified at 5uC from filtered soluble cell lysates (0.2-mm filters) using His GraviTrap affinity columns (GE Healthcare) according to manufacturer’s instructions. Application in binding buffer was followed by a washing step with 10 ml of binding buffer containing 60 mM imidazole and an elution step with 5 ml of elution buffer (50 mM HEPES-NaOH, pH 8.0; 300 mM NaCl; and 500 mM imidazole). Protein concentrations were determined by Bradford protein assay (Biorad) with BSA solutions as standard. Proteolytic activity of recombinant Deg proteases Purified recombinant proteases were incubated with an excess of b-casein in 250 mM buffer Tris-HCl, pH 7.0, supplemented with 20 mM CaCl2 for 30 min at 40uC. After reaction, proteins were analyzed by SDS-PAGE, as described below. Overexpression and purification of recombinant PsbO from Synechocystis 6803 Specific primers (59 CGCGCGGCAGCCATATGGTTGA- TAAGAGCCAGCTTAC 39and 59 GGATCCTCGAGCA- TATGTTAAACATCGGTGTCCAC 39) were designed to am- plify the psbO gene (sll0427) lacking the signal peptide from genomic DNA isolated from Synechocystis 6803. The PCR product was cloned into a pET-15b vector (Novagen) to produce a fusion protein containing an N-terminal His-tag using the In-Fusion Advantage PCR cloning kit (Clontech), following the manufac- turer’s instructions. Chemo-competent cells of the expression strains Rosetta 2 (DE3) or Rosetta-Gami 2 (DE3) (Novagen) were transformed with the plasmid and selected on LB agar containing 50 mg ml21 carbenicillin and 34 mg ml21 chloramphenicol [for Rosetta 2 (DE3) cells] or 50 mg ml21 carbenicillin, 34 mg ml21 chloramphenicol, 12.5 mg ml21 tetracyclin, and 50 mg ml21 streptomycin [for Rosetta-Gami 2 (DE3) cells]. 2–l cultures of selected colonies from each strain [7S from Rosetta 2 (DE3) cells and 2RG from Rosetta-Gami 2 (DE3) cells] were grown in LB supplemented with the corresponding antibiotics, at 30uC until OD600 = 0.5–0.6, at which point expression was induced by addition of 1 mM IPTG. Five hours after induction, cells were harvested by centrifugation at 10 000 g for 10 min at 4uC and resuspended in 30 ml of 20 mM sodium phosphate buffer, pH 8.0 containing 300 mM NaCl (buffer B). For the purification of the His-tagged PsbO, cells were broken on ice by 10 cycles of 10-s sonication with 20-s intervals of cooling, and centrifuged at 26 000 g, 1 h, 4uC. Supernatant was filtered through 0.2-mm filters and loaded onto a His GraviTrap affinity column (GE Healthcare) equilibrated in buffer B. After washing with 10 column volumes of buffer B containing 60 mM imidazole, bound proteins were eluted in 1-ml fractions with 10 column volumes of 500 mM imidazole Redox-Dependent Degradation by HhoA prepared in buffer B. Fractions were analyzed by SDS-PAGE and those containing the PsbO peak were pooled. The sample was concentrated and the buffer switched to 50 mM Tris-HCl, pH 7.5 by ultrafiltration (10-kDa cutoff, Amicon Ultra-15, Millipore). PsbO and PsbQ degradation assay The activity of Synechocystis rHhoA, rHhoB, and rHtrA proteases against reduced PsbO or PsbQ was assayed in vitro by incubating different samples isolated from spinach leaves (150 mg of purified PsbO or 100 mg of purified PsbQ) or from Synechocystis 6803 (125 mg of protein of PSII-enriched fraction isolated from the HT3 strain). The reaction mixture, with a final volume of 250 ml, contained 50 mM Tris-HCl buffer, pH 7.5; 4 mg of E. coli thioredoxin (Sigma); 3.5 mg of E. coli thioredoxin reductase (Sigma); and 1.6 mM b-NADPH (Sigma) [77]. After a pre-incubation period of 15 min at 37uC, 3 mg (16; 1:50 enzyme to substrate ratio) or 15 mg (56; 1:10) of purified recombinant HhoA, HhoB, or HtrA protease were added (0 h) and incubation proceeded at the same temperature. Samples of 45 ml were taken at 0 and 5 h, mixed with 15 ml of 46 sample buffer and heated at 95uC for 5 min. PsbO degradation in isolated PSII membrane fragments of spinach (400 mg Chl) was assessed in the absence or the presence of the reducing system as described above in a final volume of 200 ml. For the lumen fractions of spinach and Arabidopsis, the assay was performed as described in Hall et al. [20]. Polyacrylamide gel electrophoresis and immunoblotting Proteins were separated on 14% SDS-PAGE [76] and visualized by CBB staining. For immunoblotting, electrophoreti- cally separated proteins were transferred to PVDF membranes [78]. After electroblotting, the membranes were blocked using 10% (w/v) milk in PBS and incubated overnight at 4uC with a dilution of 1:10 000 (Synechocystis 6803 samples) or 1:20 000 (spinach samples) of primary anti-PsbO antibody (Agrisera, Sweden) or 1:5 000 of anti-PsbQ antibody (Agrisera, Sweden). For detection of Deg proteases, a 1:10 000 dilution of polyclonal antisera prepared in rabbits (Agrisera, Sweden) against recombi- nant HhoA, HhoB, or HtrA, purified as described above, was used for 2 h at room temperature. Antibodies directed against CP47 and NrtA were used as described previously [70]. In all cases, the membranes were probed with a 1:150 000 dilution of goat anti- rabbit secondary antibody conjugated to horseradish peroxidase (Biorad) and developed using the ECL Advance Western blotting reagents from GE Healthcare. Mass spectrometric analysis of PsbO In the CBB-stained SDS-PAGE of the lumen fraction of spinach, the identity of the band corresponding to PsbO was confirmed using mass spectrometry according to Yao et al. [79] and Jun et al. [80]. N-terminal sequence analysis of the spinach PsbO fragments N-terminal sequence analysis of the spinach PsbO fragments produced by rHhoA was carried out by the Edman degradation method at the Protein Analysis Center (PAC) (Karolinska Institute, Stockholm, Sweden). A sample of 50 mg of purified PsbO was digested with 2 mg of rHhoA in the presence of the reduced E. coli thioredoxin, as described above, for 5 h at 37uC. After SDS- PAGE, the proteins were transferred to a PVDF membrane, stained with 0.1% (w/v) CBB in 50% (v/v) methanol and the bands corresponding to the fragments were analyzed [81]. PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45713