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

Protein Expression and Purification
Rongchang ChenHui Wang

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.

References

Oct 1, 1995·Toxicon : Official Journal of the International Society on Toxinology·H F LaPenotiereJ L Middlebrook
Oct 29, 1998·Toxicon : Official Journal of the International Society on Toxinology·L A Smith
May 10, 2002·Nucleic Acids Research·David M Hoover, Jacek Lubkowski
Nov 6, 2002·Trends in Biochemical Sciences·Kathryn TurtonK Ravi Acharya
Feb 13, 2003·Journal of Molecular Biology·Andreas RummelThomas Binz
Sep 7, 2005·Protein Expression and Purification·Sapna SharmaBal Ram Singh
Nov 22, 2005·Protein Expression and Purification·Janice GilsdorfLeonard A Smith

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Citations

Jun 17, 2014·Anaerobe·G M S G MoreiraF C F Lobato
Mar 19, 2013·Toxicon : Official Journal of the International Society on Toxinology·Alexey Zdanovsky, Maria Zdanovskaia
Aug 13, 2011·Bioprocess and Biosystems Engineering·Kheirollah YariMahmood Tavallaei

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