Optimization of the BoNT/A-Hc expression in recombinant Escherichia coli using the Taguchi statistical method.
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.
Yari
Kheirollah
K
Department of Biology, Imam Hussein University, Babaei Highway, Tehran, Iran.
Fatemi
Seyed Safa-Ali
SS
Tavallaei
Mahmood
M
Tavallaei
Mahmoud
M
D000704:Analysis of Variance; D019274:Botulinum Toxins, Type A / Q000235:genetics* / Q000302:isolation & purification; D003014:Clostridium botulinum / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D007218:Industrial Microbiology / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A