The vector pET4TH used for the synthesis of the recombinant 4THase without the signal peptide was described previously (Kanao et al., 2007). Escherichia coli BL21 Star™(DE3) harboring pET4TH was cultured in a modified Terrific broth medium [90 mM potassium phosphate buffer (pH 7.2) containing 1.2% w/v tryptone, 2.4% w/v yeast extract, and 0.4% v/v glycerol] supplemented with ampicillin (50 μg mL−1) at 37 °C to an OD660 nm of 1.0. Expression of the recombinant gene was induced by adding 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to the culture, followed by incubation

at 20 °C for 36 h. Cells were harvested by centrifuging at 10 000 g for 10 min and washed three times with 100 mM of potassium phosphate buffer (KPB) (pH 7.0). The bacterial pellets were suspended in 100 mM KPB (pH 7.0) containing 2 mM dithiothreitol and disrupted by sonication on ice (the total ‘on’ period was 15 min in cycles of 30 s ‘on’ and 30 s ‘off’). The insoluble fraction INK 128 was collected by centrifugation at 10 000 g for 10 min, and the supernatant was removed. The pellet was washed three times with 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA. In order to collect the inclusion bodies, the pellet was washed with 100 mM KPB (pH 7.0) containing 4% v/v Triton X-100 three times. The inclusion Bortezomib cell line bodies were washed again

three times with sterilized distilled water to remove the detergent. The standard refolding protocol was performed as follows: recombinant proteins from the inclusion bodies were solubilized with a 6 M guanidine hydrochloride solution containing 10 mM dithiothreitol and subsequently centrifuged at 10 000 g for 10 min. The supernatant (1 mL)

containing solubilized recombinant protein was dialyzed against the refolding buffer (100 mL) at 4 °C with gentle stirring for 1 h. A solution containing 4 M guanidine hydrochloride, 10 mM β-alanine, 30% v/v glycerol, 0.4 M ammonium sulfate, and 2 mM dithiothreitol was used as the initial refolding buffer. The pH was adjusted to 4.0 with sulfuric acid. After the 1-h dialysis, the concentration of guanidine hydrochloride PI-1840 in the refolding buffer was gradually decreased by pumping the same buffer without guanidine hydrochloride into the refolding buffer using a peristaltic pump (90 s mL−1). When the volume of the refolding buffer reached 200 mL (the guanidine hydrochloride concentration was 2 M at this stage), 100 mL of the refolding buffer was removed. This dilution step was performed four times in total. When the concentration of guanidine hydrochloride in the refolding buffer was diluted to 0.25 M, the refolding buffer was replaced with a buffer (pH 4.0) containing 0.1 M β-alanine and 0.4 M ammonium sulfate. The recombinant protein solution (1 mL) was dialyzed against the buffer (1000 mL) for 3 h with gentle stirring at 4 °C. After dialysis, the dialyzed solution was centrifuged at 10 000 g for 10 min to remove insoluble proteins.