Thus, E195 and E368 (marked CX-6258 mouse with two boxes), which located in two conserved regions, were thought to be the active site residues of Gal308 based on amino acid sequence alignment and the determined structure of β-galactosidase from T. Thermophilus (Figure 1). Figure 1 Identification of the active site residues of Gal308 by alignment of the amino acid residues with other five homologous

β-galactosidases from GH family 42. The GenBank accession numbers are as follows: Geobacillus thermocatenulatus, AAW56416; Truepera radiovictrix DSM17093, ADI14846; Thermus thermophilus, ABI35985; Alicyclobacillus acidocaldarius, AAZ81841; Bacillus circulans, AAA22260; This study (Gal308), AFD21844. The alignment was carried out using the Clustal W method. The number flanking the sequences represents amino acid positions of each sequence. Asterisks mean identity. The two putative catalytic residues (E195 and E368) of Gal308 were shown in box. Heterologous expression and purification of recombinant Epigenetics inhibitor Gal308 To investigate the biochemical properties of Gal308, E. coli expression vector pET-32a(+) was used to express recombinant protein under the conditions described in materials and methods.

The cells were harvested and disrupted by sonication in ice-water bath. The cell lysate was found fully clear, and no inclusion bodies were formed, which suggested that the recombinant Gal308 was highly soluble. Then, the recombinant Lac308 with a six-histidine tag was purified by Ni-NTA chromatography, and the result showed that Ni-NTA chromatography of cell lysate led to 6.25-fold purification and 85% activity yield (Table 1). Furthermore, the purified

enzyme and the crude enzyme (supernatant from cell lysates) were applied to SDS-PAGE (Figure 2) together to determine the molecular mass and expression level of recombinant protein. The purified recombinant protein showed a single protein band of approximate 95 kDa, higher than its calculated molecular mass (76.77 kDa), which can be ascribed to its N-terminal fusion of 156 amino acids (about 18 kDa) corresponding to thioredoxin tag (Trx·Tag), polyhistidine tag (His·Tag), S·Tag epitope oxyclozanide (S·Tag), and a unique thrombin cleavage site (thrombin). In addition, the highest expression level of gal308 in E. coli was about 125 mg/L when the cell was induced at 30°C for 8 h. Next, the purified Gal308 was used to study its biochemical properties. Table 1 Purification of Gal308 Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Fold purification Activity yield (%) Cell lysate 37.94 1122.21 29.58 1.00 100.00% Ni-NTA chromatography 5.16 953.88 184.86 6.25 85.00% Figure 2 SDS-PAGE analysis of recombinant Gal308 from supernatant of E. coli BL21 (DE3) cell lysates and purified Gal308 by affinity chromatography. Lanes: M, standard protein molecular mass markers (sizes in kilodaltons are indicated on the left); 1, recombinant Gal308 from supernatant of E.