title: Improved soluble expression and characterization of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli by using a PCR-synthesized gene and a Trx co-expression strain.
abstract: Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR. The AHc was expressed at a high level in Escherichia coli successfully. Because of using the Trx co-expression strain, the expressed AHc is in a soluble and active form. The yield of the purified AHc was about 70mg/L, and its purity was up to 90% through one-step affinity chromatography. The AHc was positively identified by the antibodies raised against BoNT/A using immunological-dot-blot and Western blot assays. AHc was shown to bind with gangliosides and elicit immunity against BoNT/A, indicating that the expressed and purified AHc protein retains a functionally active conformation. Furthermore, the purified AHc has a strong immunogenicity and can be used as a potential subunit candidate vaccine for botulinum toxin serotype A.
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China.
authors.forename: Rongchang
authors.initials: R
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Shi
authors.forename: Kun
authors.initials: K
authors.lastname: Cai
authors.forename: Wei
authors.initials: W
authors.lastname: Tu
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Hou
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.forename: Le
authors.initials: L
authors.lastname: Xiao
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.forename: Yunming
authors.initials: Y
authors.lastname: Tang
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003014:Clostridium botulinum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D005260:Female; D005732:Gangliosides; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044967:Serum; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D000906:Antibodies; D005732:Gangliosides; D013879:Thioredoxins; D019274:Botulinum Toxins, Type A