Expression of Recombinant Hybrid Peptide Gaegurin4 and LL37 using Fusion Protein in E. coli
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Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1–16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.
Abstract
Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1–16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.
참고문헌 (23)
- Andersson, E, "Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes", Hum. Reprod, 10, pp.2529~2534 (2002)
- Bals, R, "Epithelial antimicrobial peptides in host defense against infection", Respir. Res, 1, pp.141~150 (2000)
- Boman, H. G, "Cell free immunity in Cecropia. A model system for antibacterial proteins", Eur. J. Biochem, 210, pp.23~31 (1991)
- Boman, H. G, "Antibacterial peptides: basic facts and emerging concepts", J. Intern. Med, 254, pp.197~215 (2003)
- Frasca, L, "Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases", Curr. Pharm. Biotechnol (2012)
- Frohm, M, "The expression of the gene coding for the antibacterial peptide LL37 is induced in human keratinocytes during inflammatory disorders", J. Biol. Chem, 272, pp.15258~15263 (1997)
- Harder, J. J, "A peptide antibiotic from human skin", Nature, 387, pp.861~862 (1997)
- Ingham, A. B., "Recombinant production of antimicrobial peptides in heterologous microbial systems", Biotechnol. Appl. Biochem, 47, pp.1~9 (2007)
- Jing, X. L, "High-level expression of the antimicrobial peptide plectasin in Escherichia coli", Curr. Microbiol, 61, pp.197~202 (2010)
- Krahulec, J, "High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli", Appl. Microbiol. Biotechnol, 88, pp.167~175 (2010)
- Kwon, S. Y, "Structural organization and expression of the gaegurin 4 gene of Rana rugosa", Biochim. Biophys. Acta, 1492, pp.185~190 (2000)
- Lai, Y, "AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense", Trends. Immunol, 30, pp.131~141 (2009)
- Miyasaki, K. T, "β-sheet antibiotic peptides as potential dental therapeutics", Int. J. Antimicrob. Agents, 9, pp.269~280 (1998)
- Nijnik, A., "The roles of cathelicidin LL-37 in immune defences and novel clinical applications", Curr. Opin. Hematol, 16, pp.41~47 (2009)
- Park, J. M., "Antimicrobial peptides from the skin of a Korean frog, Rana Rugosa", Biochem. Biophy. Res. Commun, 205, pp.948~954 (1994)
- Ramos, R, "Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum", Protein Expr. Purif, 71, pp.1~7 (2010)
- Smith, R. D, "Antimicrobial resistance: a global response", Bull. World Health Organ, 80, pp.126~133 (2002)
- Tsai, H, "Human salivary histatins: promising anti-fungal therapeutic agents", Crit. Rev. Oral Biol. Med, 9, pp.480~497 (1998)
- Turner, J., "Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils", Antimicrob. Agents. Chemother, 42, pp.2206~2214 (1998)
- Wiesner, J, "Antimicrobial peptides, The ancient arm of the human immune system", Virulence, 1, pp.440~464 (2010)
- Wilmes, M, "Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation", Nat. Prod. Rep, 28, pp.1350~1358 (2011)
- Yang, Y. H, "Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL37, in E.coli", Protein Expr. Purif, 37, pp.229~235 (2004)
- Zasloff, M, "Antimicrobial peptides of muticellular organisms", Nature, 415, pp.389~395 (2002)