Abstract
Large quantities of antimicrobial peptides are required for investigations and clinical trials, therefore suitable production method alternative to traditional chemical synthesis is necessary. Production of recombinant antimicrobial peptides in prokaryotic systems has successfully demonstrated the viability of this approach. Production of antimicrobial peptides in Escherichia coli is potentially limited due to their toxicity to host cells and susceptibility to proteolytic degradation, which can be avoided using fusion protein approach. We describe antimicrobial peptide production in E. coli based on forcing antimicrobial peptides into inclusion bodies, which is affective for the production of large quantities of antimicrobial peptides. Chemical reagents for cleaving peptide bond between antimicrobial peptides and fusion proteins such as cyanogen bromide and diluted acid are selective and provide antimicrobial peptides for biological studies in short time.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Hara, S. and Yamakawa, M. (1996) Production in Escherichia coli of moricin, a novel type antibacterial peptide from the silkworm, Bombyx mori. Biochem. Biophys. Res. Commun. 220, 664–669.
Shen, Y., Lao, X. G., Chen, Y., Zhang, H. Z., and Xu, X. X. (2007) High-level expression of cecropin X in Escherichia coli. Int. J. Mol. Sci. 8, 478–491.
Cipakova, I., Gasperik, J., and Hostinova, E. (2006) Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli. Protein Expr. Purif. 45, 269–274.
Kim, H. K., Chun, D. S., Kim, J. S., Yun, C. H., Lee, J. H., Hong, S. K., and Kang, D. K. (2006) Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli. Appl. Microbiol. Biotechnol. 72, 330–338.
Ingham, A. B. and Moore, R. J. (2007) Recombinant production of antimicrobial peptides in heterologous microbial systems. Biotechol. Appl. Biochem. 47, 1–9.
Xu, Z. N., Peng, L., Zhong, Z. X., Fang, X. M., and Cen, P. L. (2006) High-level expression of a soluble functional antimicrobial peptide, human beta-defensin 2, in Escherichia coli. Biotechnol. Progress. 22, 382–386.
Majerle, A., Kidric, J., and Jerala, R. (2000) Production of stable isotope enriched antimicrobial peptides in Escherichia coli: an application to the production of a N-15-enriched fragment of lactoferrin. J. Biomol. Nmr. 18, 145–151.
Wei, Q. D., Kim, Y. S., Seo, J. H., Jang, W. S., Lee, I. H., and Cha, H. J. (2005) Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli. Appl. Environ. Microb. 71, 5038–5043.
Hwang, S. W., Lee, J. H., Park, H. B., Pyo, S. H., So, J. E., Lee, H. S., Hong, S. S., and Kim, J. H. (2001) A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli. Mol. Biotechnol. 18, 193–198.
Lee, J. H., Kim, J. H., Hwang, S. W., Lee, W. J., Yoon, H. K., Lee, H. S., and Hong, S. S. (2000) High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies. Biochem. Biophys. Res. Commun. 277, 575–580.
Reichhart, J. M., Petit, I., Legrain, M., Dimarcq, J. L., Keppi, E., Lecocq, J. P., Hoffmann, J. A., and Achstetter, T. (1992) Expression and secretion in yeast of active insect defensin, an inducible antibacterial peptide from the fleshfly phormia-terraenovae. Invertebr. Reprod. Dev. 21, 15–24.
Andersons, D., Engstrom, A., Josephson, S., Hansson, L., and Steiner, H. (1991) Biologically-active and amidated cecropin produced in a baculovirus expression system from a fusion construct containing the antibody-binding part of protein-A. A. Biochem. J. 280, 219–224.
Rao, X. C., Li, S., Hu, J. C., Jin, X. L., Hu, X. M., Huang, J. J., Chen, Z. J., Zhu, J. M., and Hu, F. Q. (2004) A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli. Protein Expr. Purif. 36, 11–18.
Moon, J. Y., Henzler-Wildman, K. A., and Ramamoorthy, A. (2006) Expression and purification of a recombinant LL-37 from Escherichia coli. Biochim. Biophys. Acta. 1758, 1351–1358.
Zorko, M., Japelj, B., Hafner-Bratkovic, I., and Jerala, R. (2009) Expression, purification and structural studies of a short antimicrobial peptide. Biochim. Biophys. Acta 1788, 314–323.
Jonasson, P., Liljeqvist, S., Nygren, P. A., and Stahl, S. (2002) Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechol. Appl. Biochem. 35, 91–105.
Kuliopulos, A. and Walsh, C. T. (1994) Production, purification, and cleavage of tandem repeats of recombinant peptides. J. Am. Chem. Soc. 116, 4599–4607.
Lee, J. H., Skowron, P. M., Rutkowska, S. M., Hong, S. S., and Kim, S. C. (1996) Sequential amplification of cloned DNA as tandem multimers using class-IIS restriction enzymes. Genetic Anal. Biomol. Eng. 13, 139–145.
Gaussier, H., Morency, H., Lavoie, M. C., and Subirade, M. (2002) Replacement of trifluoroacetic acid with HCl in the hydrophobic purification steps of pediocin PA-1: a structural effect. Appl. Environ. Microb. 68, 4803–4808.
Kohno, T., Kusunoki, H., Sato, K., and Wakamatsu, K. (1998) A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: an application to the production of mastoparan-X uniformly enriched with N-15 and N-15/C-13. J. Biomol. Nmr. 12, 109–121.
Haught, C., Davis, G. D., Subramanian, R., Jackson, K. W., and Harrison, R. G. (1998) Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli. Biotechnol. Bioeng. 57, 55–61.
Skosyrev, V. S., Rudenko, N. V., Yakhnin, A. V., Zagranichny, V. E., Popova, L. I., Zakharov, M. V., Gorokhovatsky, A. Y., and Vinokurov, L. M. (2003) EGFP as a fusion partner for the expression and organic extraction of small polypeptides. Protein Expr. Purif. 27, 55–62.
Fassina, G., Merli, S., Germani, S., Ciliberto, G., and Cassani, G. (1994) High-yield expression and purification of human endothelin-1. Protein Expr. Purif. 5, 559–568.
Xu, X. X., Jin, F. L., Yu, X. Q., Ji, S. X., Wang, J., Cheng, H. X., Wang, C., and Zhang, W. Q. (2007) Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr. Purif. 53, 293–301.
Morassutti, C., De Amicis, F., Bandiera, A., and Marchetti, S. (2005) Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system. Protein Expr. Purif. 39, 160–168.
Wei, Q. D., Kim, Y. S., Seo, J. H., and Cha, H. J. (2005) Facilitation of expression and purification of antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli. Appl. Environ. Microbiol. 71, 5038–5043.
Moon, W. J., Hwang, D. K., Park, E. J., Kim, Y. M., and Chae, Y. K. (2007) Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin. Protein Expr. Purif. 51, 141–146.
Tang, H. Y. and Speicher, D. W. (2004) Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues. Anal. Biochem. 334, 48–61.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zorko, M., Jerala, R. (2010). Production of Recombinant Antimicrobial Peptides in Bacteria. In: Giuliani, A., Rinaldi, A. (eds) Antimicrobial Peptides. Methods in Molecular Biology, vol 618. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-594-1_5
Download citation
DOI: https://doi.org/10.1007/978-1-60761-594-1_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-593-4
Online ISBN: 978-1-60761-594-1
eBook Packages: Springer Protocols