In contrast to intracellular production, the efficient secretion of TGase or pro-TGase is considerably more cost-effective for the recovery and purification of the protein in E. coli because it does not require a cell disruption step (Mergulhao et al., 2005). In addition, secretion of

the enzyme will benefit the rapid and high throughput Dasatinib manufacturer screening of mutant libraries for desired catalytic properties. In this study, the pro-TGase from S. hygroscopicus was successfully secreted in E. coli using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was directly transformed into an active form after the addition of dispase to the culture supernatant of the recombinant strain. This is the first report of pro-TGase secretion by E. coli. In addition, we identified the residues in the pro-region of S. hygroscopicus TGase that affect the solubility and secretion of TGase in E. coli. Streptomyces hygroscopicus WSH03-13, which secretes TGase, was isolated in a previous study (Cui et al., 2007). Escherichia

coli JM109 and pMD® 19-T Simple Vector (Takara, Dalian, China) Sotrastaurin molecular weight plasmids were used for the construction of TGase-related genes. Escherichia coli BL21(DE3) and pET-22b+ (Novogen, ON, Canada) were used for the expression of pro-TGase. Streptomyces hygroscopicus genomic DNA was isolated as described previously (Kieser et al., 2000). Cloning of the TGase gene containing flanking regions from S. hygroscopicus was performed in two steps. First, the pro-TGase gene was cloned from S. hygroscopicus genomic DNA by PCR using TG-NcoI and TG-BamHI primers (Table 1) that were designed based on the conserved terminal sequence of pro-TGases from Streptomyces platensis, Streptomyces cinnamoneus, and Streptomyces fradiae (GenBank accession nos. AY555726, AB085698, and DQ432028). The target PCR product was inserted into the NcoI-BamHI sites of pET-22b+ nearly and was sequenced. Secondly, based on the sequence of the pro-TGase gene, an inverse PCR (Ochman et al., 1988) was performed to amplify the flanking regions of the cloned pro-TGase gene. Streptomyces hygroscopicus genomic DNA was digested

with PstI. The digested DNA was circularized and served as the inverse PCR template. The inverse PCR primers ITG1 and ITG2 (Table 1) were designed based on the sequence of the cloned pro-TGase gene. The PCR product containing the flanking regions of the pro-TGase gene was cloned and sequenced. Assembling the gene sequences of the pro-TGase and its flanking regions generated a TGase-related fragment that was named tgh (Fig. 1a). The signal peptide sequence prediction was performed on the signalp 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/). The promoter region sequence was predicted by bdgp (http://www.fruitfly.org/seq_tools/promoter.html). Homology searches, alignments, and other basic analyses of the nucleotide sequence were completed using vector NTI Advance 11.0 (Invitrogen, Beijing, China). A sequence-based homology model of S.