Abstract
The carboxylic domain of the Clostridium botulinum neurotoxin heavy chain (BoNT/A-HC), which has been reported as a vaccine candidate, contains the principle protective antigenic determinants. In this study, the high level expression of the BoNT/A-Hc was achieved by high cell density cultivation of recombinant Escherichia coli in a 2-l batch stirred-tank bioreactor. In order to maximize protein expression, post-induction time and IPTG inducer concentration were optimized by the Taguchi statistical design method. Results showed that the middle of the logarithmic phase and an IPTG concentration of 1 mM presented the optimum conditions for the maximum expression of BoNT/A-HC. High cell density cultivation was subsequently carried out as an effective strategy for the high level expression of recombinant BoNT/A-Hc. Consequently, soluble BoNT/A-Hc was produced at the maximum level of 486 mg l−1, at 3 h post-induction, which was approximately 9.3 and 7.8 times higher than the levels produced by the shake flask and batch culturing methods, respectively.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Zhou H, Zhou B, Johenson EA, Janda KD (2009) Selection and characterization of a human monoclonal neutralizing antibody for Clostridium botulinum neurotoxin serotype B. Bioorg Med Chem Lett 19:662–664
Yu YZ, Li N, Zhu HQ, Wang RL, Du Y, Wang S, Yu WY, Sun ZW (2009) The recombinant Hc subunit of Clostridium botulinum neurotoxin serotype A is an effective botulism vaccine candidate. Vaccine 27:2816–2822
Li L, Bal R (1999) High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain. Protein Expr Purif 17:339–344
Lin RC, Scheller RH (2000) Mechanism of synaptic vesicle exocytosis. Annu Rev Cell Dev Biol 16:19–49
Lapenotiere HF, Clayton MA, Middlebrook JL (1995) Expression of a large, nontoxic fragment of botulinum neurotoxin serotype A and its use as an immunogen. Toxicon 33:1383–1386
Tavallaie M, Chenal A, Gillet D, Pereira Y, Manich M, Gibert M, Raffestin S, Popoff MR, Marvaud JCH (2004) Interaction between two sub domain of the c-terminal part of the BON/A is essential for generation of protective antibodies. FEBS Lett 572:299–306
Pless DD, Toress ER, Reinke EK, Bavari S (2001) High-affinity, protective antibody to the binding domain of botulinum neurotoxin type A. Infect Immun 69:570–574
Chen R, Shi J, Cai K, Tu W, Hou X, Liu H, Xiao L, Wang Q, Tang Y, Wang H (2009) Improved soluble expression 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 Expr Purif 71:79–84
Choi J, Keum K, Lee S (2006) Production of recombinant proteins by high cell density culture of Escherichia coli. Chem Eng Sci 61:876–885
Durany O, Mas C, Lopez-Santin J (2005) Fed-batch production of recombinant fuculose-1-phosphate aldolase in E. coli. Process Biochem 40:707–716
Yari K, Fatemi SSA, Tavallaei M (2010) Optimization of the BoNT/A-Hc expression in recombinant Escherichia coli using Taguchi statistical method. Biotechnol Appl Biochem 56:35–42
Shojaosadati SA, Varedi Kolaei SM, Babaeipour V, Farnoud AM (2008) Recent advances in high cell density cultivation for production of recombinant protein. Iranian J Biotech 6:63–84
Babu KR, Swaminathan S, Marten S, Khanna N, Rinas U (2000) Production of interferon-α in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins. Appl Microbiol Biotechnol 53:655–660
Khalilzadeh R, Shojaosadati SA, Bahrami A, Maghsoudi N (2003) Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli. Biotechnol Lett 25:1989–1992
Vaiphei ST, Pandey G, Mukherjee KJ (2009) Kinetic studies of recombination human interferon-gamma expression in continuous cultures of E. coli. J Ind Microbiol Biotechnol 36:1453–1458
Sambrook J, Fristsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Matsui T, Sato H, Yamamuro H, Misawa S, Shinzato N, Matsuda H, Takahashi J, Sato S (2008) High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production. J Biotechnol 134:88–92
Fatemi SSA, Yakhchali B, Towfighi Darian J, Shojaosadati SA, Zomorodipour A, Karkhane AA, Rasrgar Jazii F (2003) Selection of a suitable strain from recombinant Escherichia coli strains with the same genetic structure expressing periplasmic hGM-CSF. J Biosci Bioeng 96:578–580
Liu Y, Li XL, Zhang DD, Wang Q, Liu Y, Liu DM, Wu CT, Cui CP (2010) Production of hepatopoietin Cn in high-cell-density cultures of recombinant Escherichia coli and detection of its antioxygen activity. Mol Biotechnol 47:111–119
Babaeipour V, Shojaosadati SA, Robatjazi SM, Khalilzadeh R, Maghsoudi N (2007) Over-production of human interferon-g by HCDC of recombinant Escherichia coli. Process Biochem 42:112–117
Zhang W, Potter K, Plantz AB, Schlegel LV, Smith AL, Meagher MM (2003) Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. J Ind Microbiol Biotechnol 30:210–215
Potter JK, Zhang W, Smith AL, Meagher MM (2000) Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic Yeast Pichia pastoris. Protein Express Purif 19:393–402
Acknowledgments
We would like to thank Dr Parvin Shariati and Dr Bagher Yakhchali, faculty members at the NIGEB for their useful comments.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yari, K., Fatemi, S.SA. & Tavallaei, M. High level expression of recombinant BoNT/A-Hc by high cell density cultivation of Escherichia coli . Bioprocess Biosyst Eng 35, 407–414 (2012). https://doi.org/10.1007/s00449-011-0579-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-011-0579-y