Additionally,

the pelB-mediated secretion of the precursor of a thermophilic subtilase in E. coli increased threefold after a mutation of its pro-region (Fang et al., 2010). These findings suggest that the N-terminal pro-region greatly influences protein secretion mediated by signal peptides in E. coli. Notably, the amino acid sequence PS341 homology between the pro-regions of TGases from S. mobaraensis and S. hygroscopicus is low (45.6%), whereas their mature regions shared a 79.2% homology. The pro-TGase from S. hygroscopicus may have a secretion-competent pro-region that is different from that of the pro-TGase from S. mobaraensis. The N-terminal deletions performed in this study preliminarily identified the residues in the pro-region that affect pro-TGase solubility and secretion. It was shown that the first six amino acids have an impact on pro-TGase secretion, and the next 10 residues are responsible for soluble

buy Target Selective Inhibitor Library expression. In general, a protein goes through a series of three steps before its secretion in E. coli: translocation across the cytoplasmic membrane, signal peptide cleavage in the periplasm, and translocation across the outer membrane (Mergulhao et al., 2005). Following the removal of the first six amino acids of the pro-region, TGase activity was detected in the periplasm but not in the cytoplasm after dispase treatment (data not shown), suggesting that the intracellular pro-TGase derivative (Fig. 3c) produced by the deletion was exported into the periplasm. Accordingly, the first six amino acids of the pro-region may affect pro-TGase secretion by improving its translocation across the outer membrane of E. coli. The next

10 residues (amino acids 7–16) in the pro-region contain five conserved residues (serine11, tyrosine12, alanine13, glutamic acid14, and threonine15) (Fig. 1b), and deletion of the 10 residues resulted in an insoluble pro-TGase derivative (Fig. 3d). Structural modeling of the pro-TGase showed that the five conserved residues constitute the first α-helix of the pro-region and that tyrosine12 interacts with asparagine362 and asparagine334 in the mature MG-132 datasheet region through a hydrogen bond (Fig. 4). Similar interactions between the pro-region and the mature region were also identified in the recently published crystal structure of pro-TGase from S. mobaraensis (Yang et al., 2011). During the maturation of the alpha-lytic protease precursor, the N-terminal pro-region folds into a stable structure, which acts as a scaffold for packing of the mature region into a native structure (Chen & Inouye, 2008). Therefore, it is possible that the α-helix of the pro-region assists TGase folding through a hydrogen bond interaction, and the absence of this assistance leads to the production of an insoluble pro-TGase derivative.