The use of the recombinant BoNT/A-Hc (carboxylic domain of the Clostridium botulinum neurotoxin heavy chain) has been proposed as a vaccine candidate for botulism. This fragment contains the principle protective antigenic determinant. In the present study, in order to maximize recombinant protein expression, after verification of recombinant BoNT/A-Hc by Western blotting, modified M9 medium was selected as a simple medium, and the operational and medium-composition variables together with their interactions were optimized by using the Taguchi statistical method. ANOVA for the obtained data indicated that 3.5 g, 15 g, 30 g, 15 g, 4 g, 0.7 mM, 1.5 ml per litre of medium, 30 degrees C and 15 h represented optimum values of (NH(4))(2)SO(4), glucose, K(2)HPO(4), KH(2)PO(4), MgSO(4) * 7H(2)O, isopropyl beta-D-thiogalactoside concentration, trace-elements solution, temperature and post-induction time respectively. Consequently, under these optimum conditions, 52.1 mg/l of soluble BoNT/A-Hc was obtained in shake flask culture.