br Materials and methods br

Materials and methods
Results
Discussion D. abbreviatus larvae are voracious feeders, and the nutrients consumed as part of their diet are broken by gut proteases and utilized for growth and development. Since proteases play an important role in larval and adult food digestion, it is important to characterize these proteases in order to be able to effectively control them in future crop protection strategies. Numerous proteases are transcribed during larval feeding stages and the majority of these proteases belong to the serine (chymotrypsins, trypsins and serine carboxypeptidases) and cysteine (cathespsins B and L) protease families (Table 2) that are common in Coleoptera (Thie and Houseman, 1990, Hernández et al., 2003, Edwards et al., 2010). Of the cysteine proteases, cathepsin L1 (Da-CTSL1) is encoded by the most abundant transcript that comprises 64% of the total cathepsin transcripts considering the fact that 24 different members of this gene superfamily were identified, and, therefore, Da-CTSL1 is probably important in food digestion in the gut of 5week old larvae (Fig. 1A). Assaying segments of the larval gut by RT-qPCR show that Da-CTSL1 transcripts are 1000-fold more abundant in the foregut and the anterior midgut than in the posterior mid gut where the pH is basic (Fig. 1B). These findings strongly indicate that even though the pro-enzyme is synthesized by the foregut and anterior midgut (Fig. 1C) it is not activated until it reaches the posterior midgut which is basic (Fig. 1B). To better understand its significance, we cloned, expressed and characterized Da-CTSL1. Several expression systems were used to express cathepsin L in vitro. Bown et al. (2004) and Fonseca et al. (2012) have expressed cathepsin L from the gut of the western corn rootworm (D. virgifera) and the sugarcane weevil, Sphenophorus levis using the methanotropic yeast P. pastoris. The procathepsin L was secreted by the ONO-5046 into the medium during the fermentation of the cells. Bown et al. (2004) reported that the secreted proenzyme was cleaved by the yeast proteases and was found as the active cathepsin L, whereas Fonseca et al. (2012) reported that the secreted S. levis proenzyme was not activated during the secretion into the medium. Ishidoh and Kominami (1994) expressed procathepsin L in v-Ha-ras transformed NIH3T3 cells and Yamamoto et al. (1999) expressed Bombyx mori preprocathepsin L in E. coli. To study the activation process at the molecular level and to avoid precipitation of the recombinant protein in the bacterial inclusion bodies Yamamoto et al. (1999) used lengthy renaturation steps recommended by Smith and Gottesman (1989). We expressed the recombinant Da-proCTSL1 in Rosetta competent E. coli cells as a fusion product with a GST tag (Fig. 4). Even though the recombinant Da-GST-proCTSL1 did not precipitate in the bacterial inclusion bodies, the recombinant Da-CTSL1 was unstable in solution and tended to precipitate upon storage at 4°C. In the future other polypeptide moieties could perhaps be fused to the enzyme to keep it soluble and retain its activity, such as the maltose binding protein (MBP) and the small ubiquitin-like modifier (SUMO) (Kapust and Waugh, 1999, Butt et al., 2005). It is also possible that because Da-CTSL1 is active at a basic pH unlike all other cathepsins that are active at acidic pH and lose activity at basic pH, Da-CTSL1 may not be stable and even precipitate at low or neural pHs. To overcome the insolubility of Da-CTSL1, Triton X-100 (1%) was added to the enzyme keeping Da-CTSL1 in solution without interfering with its protease activity (Fig. 8, Fig. 10). Although numerous alternative recombinant protein synthesis technologies are available (Hunt, 2005), expressing heterologous proteins in E. coli is simple and straight-forward. Bacteria provide the cheapest and fastest recombinant protein synthesis technology that can be easily scaled up.