title: Expression and biochemical characterization of light chains of Botulinum neurotoxin subtypes F5 and F7.
abstract: Botulinum neurotoxins are the most potent protein toxins known to human. To date, seven subtypes of the BoNT/F serotype (BoNT/F1 to BoNT/F7) have been identified, among which BoNT/F5 and BoNT/F7 are the most divergent. However, little structural and functional information is available for these two subtypes due to a lack of suitable recombinant proteins for biochemical characterization, except that they appear to possess unique substrate recognition mechanisms, thereby impeding development of vaccine or inhibitors against these proteins. In the present study, we utilized a combinatorial approach which involved examining the effects of different affinity tags, mapping C-terminal truncation mutants and optimization of expression and purification conditions, that allowed us to successfully express and purify soluble and highly active recombinant LC/F5 and LC/F7 proteins. GST-LC/F5(1-450) and 6× His-LC/F5(1-405) were the formats which exhibit the highest level of solubility and activity, whereas GST-LC/F7(1-405) was the most active form of LC/F7. In comparison, GST-LC/F5(1-450) was more active than GST-LC/F7(1-405), which was in turn more active than the LC/F1 control. Our data suggest that solubility of these proteins strongly correlated with their catalytic activity. Successful expression and purification of LC/F5 and LC/F7 in this work will, for the first time, provide materials for further characterization of these two subtypes of BoNT/F, which is essential for future development of protective vaccine or other therapeutic strategies, as well as BoNT/F protein engineering.
authors: Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shenzhen Research Institute, Shenzhen, PR China; State Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.
authors: Jiubiao
authors: J
authors: Guo
authors: Shenzhen Key Lab for Food Biological Safety Control, Food Safety and Technology Research Center, Hong Kong PolyU Shenzhen Research Institute, Shenzhen, PR China; State Key Lab of Chirosciences, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong. Electronic address: sheng.chen@polyu.edu.hk.
authors: Sheng
authors: S
authors: Chen
mesh_terms: D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D006801:Humans; D009154:Mutation
chemical_list: D001905:Botulinum Toxins; C096321:botulinum toxin type F
keywords: Biochemical characterization; Botulinum neurotoxin serotype F; Expression; F5; F7; Light chain