*2.3. Recombinant Protein Purification*

LiRecTCTP was purified by a ffinity chromatography using Ni<sup>+</sup>2-NTA column (Invitrogen) as described by Sade and colleagues [2], with modifications. Briefly, *Escherichia coli* cells were lysed by thaw–freeze cycles and disrupted by cycles of sonication. The cell lysate was centrifuged (20,000 × *g*, 20 min, 4 ◦C), and the supernatant was incubated with 1 mL Ni2<sup>+</sup>-NTA beads for 1 h at 4 ◦C. The recombinant protein was washed with wash buffer (50 mM sodium phosphate pH 6.0, 500 mM NaCl, 35 mM imidazole, Sigma Aldrich), eluted with elution buffer (50 mM sodium phosphate pH 8.0, 500 mM NaCl, 350 mM imidazole), and analyzed by 12.5% SDS-PAGE under reducing conditions. Wash buffer pH was important to the astringent condition for protein binding to the column.

### *2.4. Protein Quantification and Gel Electrophoresis*

The total protein concentration of different samples and, especially, the purified LiRecTCTP were determined by the Coomassie Blue method (BioRad, Hercules, CA, USA) [14]. For protein quality analysis, 12.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed under reducing conditions.
