coli BL21(DE3)pLysS. The transformant was grown in Luria–Bertani broth containing ampicillin (50 μg mL−1) and chloramphenicol (34 μg mL−1) Selumetinib with shaking (230 r.p.m.) at 37 °C until an OD600 nm of 0.6 was attained. The cultures were induced by adding 0.4 mM isopropyl-1-thio-d-galactopyranoside and cultivated for another 4 h. Bacterial pellets harvested by centrifugation were stored overnight at −20 °C and were then suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.2 mg mL−1 lysozyme and 0.1 mg mL−1

DNAse. Cells were disrupted by sonication, and subsequent centrifugation (30 min at 16 000 g) allowed the collection of inclusion bodies containing the recombinant T. cervina LiP proteins. Trametes cervina LiP proteins that were either isolated from the culture medium (Miki et al., 2006) or heterologously produced in E. coli were purified using sodium dodecyl sulfate polyacrylamide gel electrophoresis. In-gel tryptic digestion and matrix-assisted laser desorption/ionization-time-of-flight-MS (MALDI-TOF-MS) analysis were performed as described by Shimizu et al. (2005). The appropriate bands were excised. The gel pieces were washed with 40% 1-propanol and then with 0.1 M ammonium bicarbonate containing

50% acetonitrile. The proteins in the gel pieces were digested overnight with 20 ng μL−1 modified trypsin (Promega) in 0.1 M ammonium bicarbonate at 37 °C. The digested peptides were extracted with 0.1 M Cyclopamine ammonium bicarbonate and then

with 80% acetonitrile containing 0.1% trifluoroacetic acid. The extracts were combined and concentrated. The peptide solutions were analyzed by MALDI-TOF-MS (Voyager DE; Applied Biosystems) using α-cyano-4-hydroxycinnamic mTOR inhibitor acid in H2O/acetonitrile (1 : 1) containing 0.1% trifluoroacetic acid as the matrix. Some peptides were sequenced with electrospray ionization-MS (ESI-MS)/MS (Q-Tof2; Micromass). Homology modeling of T. cervina LiP was performed using the molecular operating environment (moe) program (Chemical Computing Group). The P. chrysosporium LiP crystal structure (PDB entry 1LLP) was selected as the best template due to the highest degree of amino acid sequence identity (50.1%) to T. cervina LiP. After modeling and energy minimization using the AMBER89 force field, 10 model intermediates were generated. The best intermediate with the lowest packing score (−2.2551) was used for further revision and full energy minimization: the cis-conformation at Ser300 was revised to trans-conformation using the AMBER89 force field, and full-energy minimization was run with the Engh–Huber force field. Finally, a model with a favorable geometry (root mean square deviation of Cα topology=0.008 Å) was obtained. All modeling and energy minimization steps were carried out under the conditions including heme from the template. To better understand the T. cervina LiP molecule, we isolated its cDNA and characterized its molecular structure.