The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1 similar to E11, have been identified. The LCs of BoNT/E were insoluble, prohibiting studies towards understanding the mechanisms of toxin action and substrate recognition. In this work, the molecular basis of insolubility of the recombinant LCs of two representative subtypes of BoNT/E, E1 (Beluga) and E3 (Alaska), was determined. Hydrophobicity profile and structural modeling predicted a C-terminal candidate region responsible for the insolubility of LC/Es. Deletion of C-terminal 19 residues of LC/E(1-400) resulted in enhanced solubility, from 2 to similar to 50% for LC/E-Alaska and from 16 to similar to 95% for LC/E-Beluga. In addition, resides 230-236 were found to contribute to a different solubility level of LC/E-Alaska when compared to LC/E-Beluga. Substituting residues (TCI232)-T-230 in LC/E-Alaska to the corresponding residues of (KYT232)-K-230 in LC/E-Beluga enhanced the solubility of LC/E-Alaska to a level approaching that of LC/E-Beluga. Among these LC/Es and their derivatives, LC/E-Beluga 1-400 was the most soluble and stable protein. Each LC/E derivative possessed similar catalytic activity, suggesting that the C-terminal region of LC/Es contributed to protein solubility, but not catalytic activity. In conclusion, this study generated a soluble and stable recombinant LC/E and provided insight into the structural components that govern the solubility and stability of the LCs of other BoNT serotypes and Tetanus toxin. (C) 2015 Elsevier Inc. All rights reserved.