(20 mM Tris-HCl pH 7.5 containing 500 mM NaCl, 50 mM imidazole and 1 mM TCEP), and lysed by sonication. Cell debris was removed by centrifugation at 130006 g for 30 min, and the supernatant was loaded onto a 5 mL HiTrapTM IMAC Column (GE Healthcare) charged with Ni (II) ion. Column was pre- equilibrated with the lysis buffer. After extensive washing with the lysis buffer, His-tagged protein was eluted with 300 mM imidazole in lysis buffer. Eluent was supplemented with 0.5 mg of His-tagged SUMO protease per 1 L of culture and dialyzed in 20 mM Tris- HCl pH 7.5 containing 200 mM NaCl and 1 mM TCEP at 4uC overnight. After proteolytic cleavage, the His-SUMO tag was removed by nickel affinity chromatography. The column was washed with 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 50 mM imidazole and 1 mM TCEP. Desired tag-free HypA or HypB was found in the flow-through and washing fractions. It was concentrated to a concentration of 10 mg/mL, and loaded onto a HiLoad 26/60 Superdex 75 column (GE Healthcare) equili- brated with 50 mM Tris-HCl buffer pH 7.5, 0.2 M NaCl and 1 mM TCEP. Eluted fractions were analyzed by SDS-PAGE. Purified HypA was eluted at elution volume of ,205 ml and purified HypB was eluted at elution volume of ,165 ml. Apo- AfHypB was prepared by extensive dialysis in 50 mM Tris-HCl buffer pH 7.5, 0.2 M NaCl, 1 mM TCEP and 5 mM EDTA to remove the bound metal and nucleotide [31]. EDTA was later removed by dialysis again. Purified proteins were snap frozen with liquid nitrogen, and stored in 280uC deep freezer before use. Determination of molecular weight of AfHypB preloaded with GDP, GMPPNP with and without nickel by size- exclusion-chromatograpghy/light-scattering (SEC/LS) AfHypB (0.5 mM) was mixed with 5 mM GMPPNP or GDP, with or without 1 mM NiCl2. After removal of excess ligands by gel filtration, the AfHypB samples were loaded to a Superdex-75 HR10/30 column (GE Healthcare) equilibrated with 50 mM Tris pH 7.5, 0.2 M NaCl, 5 mM MgCl2 and 1 mM TCEP. The molecular weight of the AfHypB samples was measured by a downstream multiple-angle laser light scattering detector (Mini- DAWN light scattering detector, Wyatt Technology). Data were analyzed using Astra version 5.3.4.18 (Wyatt Technology), following the manufacturer’s instructions. Determination of molecular weight of AfHypA/AfHypB complex The molecular weight of A. fulgidus HypA/HypB complex was determined using SEC/LS. Protein samples were analyzed by loading onto a Superdex-75 HR10/30 column (GE Healthcare) equilibrated with the 50 mM Tris pH 7.5, 0.2 M NaCl and 1 mM TCEP, and eluted at room temperature at a flow rate of 0.5 mL/ min. The column was connected downstream to a multiple-angle laser light scattering (Mini-DAWN light scattering detector, Wyatt Technology). Data were analyzed using Astra version 5.3.4.18 (Wyatt Technology), following the manufacturer’s instructions. GST pull-down assay Interaction between AfHypA and AfHypB was tested by GST pull-down assay using GST-AfHypA as a bait. 50 mL overnight culture of E. coli cells expressing GST-AfHypA and His-SUMO- AfHypB were collected by centrifugation and lysed by sonication in 5 mL phosphate buffered saline (PBS). To remove the His-SUMO tag, 0.5 mg of SUMO protease was added to the clear cell lysate of His-SUMO-AfHypB. After incubation at 25uC for 1 hour, the lysates containing AfHypB and GST-AfHypA were mixed together and loaded onto 0.1 mL glutathione(GSH)-resin. After extensive HypA/HypB Interaction and Hydrogenase Maturation washing PBS, bound proteins were eluted with 20 mM Tris pH 8.0, 0.1 M NaCl and 20 mM reduced GSH. The eluted proteins were analyzed by a 15% SDS-PAGE stained with Coomassie blue. The GST pull-down assay for EcHypA and EcHypB was performed as described for AfHypA and AfHypB with the exception that the His-SUMO tag of EcHypB was not cleaved so that the bands of GST-EcHypA and His-SUMO-HypB can be clearly distinguished in SDS-PAGE analysis Hydrogenase reactivation assay Hydrogenase reactivation from crude cell extract was measured by a modified method based on the procedures of Ballantine and Boxer [32] and Zhang et al. [33]. The E. coli DhypB strain (F2, 2 D(araD-araB)567, DlacZ4787 (::rrnB-3), l , DhypB731::kan, rph-1, D(rhaD-rhaB)568, hsdR514) (Keio collection no.: JW2697-1), obtained from the Keio collection, E. coli Genetic Stock Center [34], was transformed with the wild-type pBAD-EcHypB or its mutants. The DhypB genotype and the pBAD-EcHypB plasmid were selected by 50 mg/ml kanamycin and 100 mg/ml ampicillin, respectively. Starter culture was grown from single colonies in LB medium at 37uC. After 2% (v/v) inoculation, the E. coli cells were grown anaerobically using 50 mL sealed plastic tubes in buffered TGYEP medium (10 g/L tryptone, 5 g/L yeast extract, 0.8% glycerol, 69 mM K2HPO4, and 22 mM KH2PO4) supplemented with 15 mM sodium fumarate, 1 mM sodium molybdate, and 1 mM sodium selenite for 16 hours. The expression was induced by adding 1 mM arabinose. Cells were harvested by centrifugation and washed with 50 mM potassium phosphate buffer, pH 7.0, and resuspended in the same buffer containing 1 mM dithiothreitol and 0.2 mM phenylmethylsulfonyl fluoride. Crude cell extracts were prepared by sonication and subsequent centrifugation at 13,0006 g for 30 min at 4uC. The protein concentrations of crude cell extracts were determined by the Bradford protein assay kit (BioRad) using bovine serum albumin as standard. Total hydrogenase activity of the crude cell extracts was measured by hydrogen-dependent reduction of benzyl viologen. Reactions were prepared in a septum-sealed cuvette. Sealed cuvette containing 2.5 mL of 50 mM potassium phosphate, pH 7.0 was degassed thoroughly by vacuum pump followed by bubbling with 95% N2 and 5% H2 for 15 minutes. 200 mL solution of 0.05% (w/v) sodium dithiolate was injected into the cuvette to remove the remaining oxidative species. To start a reaction, 300 mL of degassed diluted crude cell extract (at 0.2 mg/ mL of total protein) was injected into the cuvette. The initial OD600 should be in the range of 0.2–0.5 to ensure there was no remaining oxidative species in the reaction. OD600 was monitored at 25uC for 15 minutes. The amount of benzyl viologen reduced was quantified by changes in OD600 using the extinction coefficient of 7400 M21 cm21. Hydrogenase activity was measured as nanomol of benzyl viologen reduced per min per mg of total protein. Activity measurement of each condition was repeated for three times in individual experiment. Hydrogenase activity of the wild type parental strain E. coli (F-, D(araD-araB)567, 2 DlacZ4787(::rrnB-3), l , rph-1, D(rhaD-rhaB)568, hsdR514) trans- formed with pBAD-A was measured along with each batch of reaction as positive control and relative standard. Supporting Information Table S1 Constructs and Primers used. Desired DNA fragment was amplified by PCR the from genomic DNA using the corresponding primer pair, followed by restriction digestion to create compatible ends on both inserts and plasmids. DNA ligation was performed to generate the desired plasmid. Restriction PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e32592