To investigate the effect of pyrroloquinoline quinone (PQQ) (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan) for the protein refolding, PQQ at the concentration of 70 μM was added to both the refolding and the dialysis buffers. The reaction mixture (1 mL) contained 2 mM K2S4O6, 200 mM K2SO4, 100 mM β-alanine nitrate buffer (pH 3.0), and 50 μL of enzyme solution in thin glass tubes.

In the case of purified enzyme, the protein concentration of the solution was 0.1 mg mL−1. The reaction was initiated by adding 50 μL of enzyme solution at 30 °C. After incubating for the appropriate reaction period, the reaction tubes were immediately placed in cold ethanol (−20 °C) with shaking HIF inhibitor for 2 min, followed by boiling for 90 s to stop the reaction. Because elemental sulfur was produced by the reaction, the samples were centrifuged

at 10 000 g for 1 min to remove the byproduct (S0). The enzyme activity was measured by determining the concentration of tetrathionate remaining in the supernatant by cyanolysis (Nor & Tabatabai, 1975). One unit (U) of the activity was defined as the amount of enzyme required for the hydrolysis of 1 μmol tetrathionate min−1. Quinoproteins were detected by staining with nitroblue tetrazolium (NBT) (Paz et al., 1991; TGF-beta inhibitor Rzhepishevska et al., 2007). The NBT solution contained 0.24 mM NBT in 2 M potassium glycinate buffer (pH 10). Protein samples were blotted onto a nitrocellulose membrane and dried at room temperature. The membrane was immersed in the NBT solution for 45 min in the dark, and subsequently dipped into 0.1 M sodium borate (pH 10). Quinoproteins could be specifically detected as purple-blue spots due to NBT reduction to formazan. PQQ (Mitsubishi Gas Chemical Company Inc.), used as a positive control and blotted onto a nitrocellulose membrane, was treated as described above. The plasmid pET4TH encoding the recombinant 4THase without the signal peptide was introduced 4��8C into

E. coli BL21 Star™(DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the soluble and insoluble fractions revealed the presence of a recombinant protein in the insoluble fraction (Fig. 1, lane 2), suggesting that the protein was synthesized in inclusion bodies. Some proteins derived from host cells were removed by washing the inclusion bodies with the solution containing Triton X-100. As shown in Fig. 1, the recombinant protein prepared from the washed inclusion bodies exhibited a single main band on SDS-PAGE (Fig. 1, lane 3). The refolding experiments of recombinant Af-Tth synthesized in inclusion bodies of E. coli were carried out to obtain the recombinant Af-Tth in the active form. The protein solubilized from inclusion bodies with 6 M guanidine hydrochloride solution containing 10 mM dithiothreitol was used for the refolding experiments. Initially, the effect of pH on the refolding of recombinant Af-Tth was evaluated.