, 1998). It has been shown that expression of disulfide rich peptides in ORIGAMI (DE3) strain substantially improve the yield of active proteins purified ( Prinz et al., 1997). Only part of the recombinant PnTx3-4 was expressed as a soluble protein. The yield of Rapamycin price soluble PnTx3-4 after all the purification steps ranged from 0.5 to 0.8 mg/L, which is in the same range to what has been reported for

other animal toxins successfully expressed in E. coli ( Johnson et al., 2000; Meng et al., 2011; Che et al., 2009; Souza et al., 2008; Carneiro et al., 2003). More importantly, the soluble recombinant protein showed biological activity very similar to the native PnTx3-4, both in the glutamate release assay as well as in the measurement of intrasynaptosomal free calcium concentration.

These results indicate that, similar to the native peptide, soluble recombinant PnTx3-4 is able to block Ca2+ channels involved in glutamate release from cortical synaptosomes. Because most of Alectinib purchase the recombinant PnTx3-4 aggregated as inclusion bodies we also searched for conditions to provide efficient refolding of the insoluble recombinant PnTx3-4. Finding the exact conditions to renature proteins is usually time-consuming as refolding conditions for individual proteins vary considerably (Singh and Panda, 2005; Lilie et al., 1998). The basic protocol requires that purified inclusion bodies are first solubilised with a strong denaturant, such as guanidine hydrochloride (GdnHCl), to produce a completely unfolded protein. DTT is also added to allow reduction of disulfide bridges (Fahnert et al., 2004). The solubilised protein is then diluted or dialyzed into a refolding buffer to reduce the denaturant concentration, allowing the protein to refold based on the information contained in its primary sequence. As the denaturant is removed, protein aggregation tends to compete with renaturation therefore, it is crucial to identify the ideal milieu to recover maximal amounts of native protein. Several factors

influence renaturation/aggregation during BCKDHB refolding including protein concentration, concentration of strong and weak denaturants, pH, temperature, and the redox environment (Fahnert, 2004; Lilie et al., 1998). Out of 9 different buffer conditions (Table 3) that we tried, only buffer 5, which contained 0.5 M Gnd-HCl, 0.4 M l-arginine, 1 mM GSH and 1 mM GSSG, allowed proper refolding of PnTx3-4. Using buffer 5 we managed to obtain 1.5–2.0 mg/L of PnTx3-4 refolded after purification from inclusion bodies. Importantly, the refolded peptide also showed biological activity very similar to the native peptide. These results indicate that a balanced molar ratio of reduced to oxidized thiol reagents (glutathione) was essential to provide the appropriate redox potential to allow formation and reshuffling of disulfide bonds (Misawa and Kumagai, 1999; Wetlaufer et al., 1987).