The model was viewed, and figures were prepared using pymol (DeLano Scientific, San Carlos, CA). To construct the plasmid encoding pro-TGase containing the pelB signal peptide, the pro-TGase gene was amplified from S. hygroscopicus genomic DNA using the primer pair PTG1 and PTG2 (Table 1). To construct the plasmid

encoding pro-TGase with its endogenous signal peptide, the complete open reading frame (ORF) of the TGase gene was amplified from S. hygroscopicus genomic DNA using the primer pair ORFTG1 and ORFTG2 (Table 1). Each amplified PCR product was cloned into the NcoI-XhoI sites of pET-22b+ to produce pBB1-1010 and pBB1-1020, respectively. Each gene fragment of pro-TGase containing an N-terminal deletion was amplified from pBB1-1020 by PCR using a specific forward primer and a constant reverse Dabrafenib primer (TG2) (Table 1). For the deletion of the first six N-terminal amino acids in the pro-region, www.selleckchem.com/products/PD-0325901.html TG7 (Table 1) was used as a forward primer. For further deletions in the pro-region, TG17, TG23, TG33, and TG58 were used as the forward primers (Table 1). The resulting PCR products were inserted into

the NcoI-XhoI sites of pET-22b+ to produce pBB1-1011, pBB1-1012, pBB1-1013, pBB1-1014, and pBB1-1015, respectively (Fig. 2a). Pro-TGase and its derivatives were expressed in E. coli BL21(DE3). A seed culture of each recombinant strain was prepared by growing cells in Luria–Bertani medium containing ampicillin (100 μg mL−1) at 37 °C for 12 h. The seed culture was inoculated into Terrific Broth medium containing ampicillin (100 μg mL−1) and cultivated at 37 °C until the optical density at 600 nm reached 1.0–1.5. Isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.4 mM. After incubation for 40 h at 20–37 °C, the cells and its culture supernatant were separated by centrifugation. Cells (1 OD600 nm unit) were

sonicated in 100 μL Tris–HCl buffer (pH 8) and centrifuged. The supernatant of the sonicated cells is the intracellular soluble fraction. The cell debris from the centrifugation step was resuspended in the Tris–HCl buffer containing 1% and corresponds to the intracellular Idoxuridine insoluble fraction. The pro-TGase activation by dispase (Worthington, Lakewood, NJ) was performed as previously described (Marx et al., 2008) with the following modification. Instead of activation in the specific buffer, the activation here was initiated by directly adding dispase solution (Marx et al., 2008) to the culture supernatant of each recombinant E. coli strain. Purification of pro-TGase and TGase from S. hygroscopicus and pro-TGase from the recombinant strains was performed as previously described (Zhang et al., 2008b). Tests of TGase activity, protein content, and SDS-PAGE were conducted as previously described (Zhang et al., 2008b). Amino acid sequencing of the TGase N-terminal was performed by Shanghai Gene Core Biotechnologies Co., Ltd.