The NADPH-dependent carbonyl reductase S1 from stereoselectively catalyzes the reduced amount of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (and purified by Ni-affinity ion-exchange and size-exclusion chromatography. a novel enzyme that produces (Rosetta (DE3) (Novagen) cells were transformed with the expression vector constructed above and the recombinant cells were cultured in 1?l LB medium containing 10?μg?ml?1 kanamycin at 310?K until the OD600 reached 0.6. Protein expression was induced by the addition of 0.1?misopropyl β-d-1-thiogalactopyranoside. Cell growth was continued at 298?K for 20?h after initiating IPTG induction. The cells were harvested by centrifugation at 5000for 10?min IL3RA and were resuspended in 50?mTris-HCl buffer pH 8.0 containing 100?mNaCl and 1?mphenylmethylsulfonyl fluoride. The protein was extracted from the cells by sonication and was separated from in-soluble materials by centrifugation at 40?000for 30?min. The supernatant made up of 6×His-tagged carbonyl reductase S1 was loaded onto an Ni-Sepharose 6 Fast Flow column (GE Healthcare). After washing the resin with 100?mTris-HCl ASA404 buffer pH 7.5 made up of 1?NaCl and 40?mimidazole carbonyl reductase S1 was eluted in a single step with 50?mTris-HCl buffer pH 7.5 made up of 500?mNaCl and 500?mimidazole. The eluate was dialyzed against 20?mTris-HCl buffer pH 8.0 and the 6×His-tagged carbonyl reductase S1 was digested by thrombin protease (3?U per milligram of protein; GE Healthcare) at 277?K for 18?h. More than 95% of the protein was digested. After centrifugation at 23?000for 10?min the supernatant was applied onto a Resource Q column (GE?Healthcare) and eluted with a linear gradient of 0-400?mNaCl in 20?mTris-HCl buffer pH 8.0. Two major peaks were obtained in?the anion-exchange chromatography at around 150?mNaCl. The first peak contained carbonyl reductase S1 with and without 6×His tag (theoretical pI of 6.95 and 6.54 respectively) and the second peak exclusively contained the protein without 6×His tag. The fractions made up of carbonyl reductase S1 exclusively without 6×His tag were applied onto a Superdex 200 column (GE Healthcare) equilibrated with 20?mTris-HCl buffer pH 8.0 containing 100?mNaCl and eluted as a single peak. The chromatographic actions described above were sufficient to purify the protein (Fig. 1 ? Tris-HCl buffer pH 8.0 containing 100?mNaCl using a Vivaspin 20 centrifugal concentrator (10?kDa molecular-weight cutoff; Sartorius). The purified protein answer was cooled in liquid nitrogen and stored at 193?K. Samples were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R250 staining. Protein concentrations were calculated from the absorbance at 280?nm using a molar extinction coefficient of 39?880?Li2SO4 0.1 cacodylate pH 6.5 was obtained from Wizard I ASA404 and II (Emerald BioSystems) by the sitting-drop vapour-diffusion method in 96-well plates (Art Robbins Instruments) using purified protein solution supplemented with 5?mβ–NADPH (Oriental Yeast). The crystallization condition was optimized by varying the precipitant concentration and pH and by using Additive Screen HT (Hampton Research). The final crystallization condition was 31%(Li2SO4 0.08 potassium tartrate tetrahydrate 0.1 cacodylate pH 6.5 at 293?K using the sitting-drop vapour-diffusion method in 24-well plates (Hampton Research). The crystallization drops were prepared by mixing 1?μl protein solution supplemented with 5?mβ-NADPH and 1?μl reservoir solution and were equilibrated against 0.5?ml reservoir solution. The crystals grew to dimensions of 60 × 60 × 200?μm within 3?d. Fig. 2 ? shows ASA404 the crystals obtained using the final crystallization condition. Physique 2 Common crystals of carbonyl reductase S1 at 293?K obtained using the optimized crystallization conditions. The optimum amount of the crystals was 0 approximately.2?mm. Crystals had been soaked within a 7:3 combination of tank option and ASA404 100% ethylene glycol. The crystals had been mounted in nylon loops (Hampton Research) and flash-cooled in a stream of nitrogen gas at 95?K. X-ray diffraction data were collected with an ADSC Quantum 210 CCD detector using synchrotron radiation around the AR-NW12 beamline of the Photon Manufacturing plant Tsukuba Japan. A total of 900 diffraction images were collected at a wavelength of 1 1.0000?? with a?crystal-to-detector distance of 189.7?mm 0.2 oscillation and.