Abstract
The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.
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Agerberth B, Charo J, Werr J, Olson B, Idali F, Lindbom L, Kiessling R, Jornvall H, Wigzell H, Gudmundsson GH (2000) The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte populations. Blood 96:3086–3093
Bals R, Wilson JM (2003) Cathelicidins—a family of multifunctional antimicrobial peptides. Cell Mol Life Sci 60:711–720
Bowdish DM, Davidson DJ, Hancock RE (2006) Immunomodulatory properties of defensins and cathelicidins. Curr Top Microbiol Immunol 306:27–66
Dorschner RA, Pestonjamasp VK, Tamakuwala S, Ohtake T, Rudisill J, Nizet V, Agerberth B, Gudmundsson GH, Gallo RL (2001) Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus. J Invest Dermatol 117:91–97
Ganz T (2004) Defensins: antimicrobial peptides of vertebrates. C R Biol 327:539–549
Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Hong IP, Lee SJ, Kim YS, Choi SG (2007) Recombinant expression of the human cathelicidin (hCAP18/LL-37) in Pichia pastoris. Biotechnol Lett 29:73–78
Horn U, Strittmatter W, Krebber A, Knüpfer U, Wenderoth R, Müller K, Matzku S, Plückthun A, Riesenberg D (1996) High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Appl Microbiol Biotechnol 46:524–532
Isogai E, Isogai H, Matuo K, Hirose K, Kowashi Y, Okumuara K, Hirata M (2003) Sensitivity of genera Porphyromonas and Prevotella to the bactericidal action of C-terminal domain of human CAP18 and its analogues. Oral Microbiol Immunol 18:329–332
Koczulla R, von Degenfeld G, Kupatt C, Krotz F, Zahler S, Gloe T, Issbrucker K, Unterberger P, Zaiou M, Karl A, Raake P, Pfosser A, Boekstegers P, Welsch U, Hiemstra PS, Vogelmeier C, Gallo RL, Clauss M, Bals R (2003) An angiogenic role for the human peptide antibiotic LL-37/hCAP-18. J Clin Invest 111:1665–1672
Lehrer RI, Granz T (1998) Antimicrobial peptides in mammalian and insect host defence. Curr Opin Immunol 11:23–27
Lehrer RI, Barton A, Ganz T (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J Immunol Methods 108:153–158
Lehrer RI, Rosenman M, Harwing SSSL, Jackson R (1991) Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 137:167–173
Li Y, Li X, Wang G (2006) Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies. Protein Expr Purif 47:498–505
Li Y, Li X, Li H, Lockridge O, Wang G (2007) A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation. Protein Expr Purif 54:157–165
Maguin E, Prevost H, Ehrlich SD, Gruss A (1996) Efficient insertion mutagenesis in Lactococci and other Gram-positive bacteria. J Bacteriol 178:931–935
Moon JY, Henzler-Wildman KA, Ramamoorthy A (2006) Expression and purification of a recombinant LL-37 from Escherichia coli. Biochim Biophys Acta 1758:1351–1358
Ong PY, Ohtake T, Brandt C, Strickland I, Boguniewicz M, Ganz T, Gallo RL, Leung DY (2002) Endogenous antimicrobial peptides and skin infection in atopic dermatitis. N Engl J Med 347:1151–1160
Oren Z, Lerman JC, Gudmundson GH, Agerberth B, Shai Y (1999) Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molar basis for its non-cell-selective activity. Biochem J 341:501–513
Schaller-Bals S, Schulze A, Bals R (2002) Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am J Respir Crit Care Med 165:992–995
Schittek B, Hipfel R, Sauer B, Bauer J, Kalbacher H, Stevanovic S, Schirle M, Schroeder K, Blin N, Meier F, Rasner G, Garbe C (2001) Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat Immunol 2:1133–1137
Shaykhiev R, Beisswenger C, Kandler K, Sneske J, Puchner A, Damm T, Behr J, Bals R (2005) Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure. Am J Physiol Lung Cell Mol Physiol 289:L842–L848
Smeianov V, Scott K, Reid G (2000) Activity of cecropin P1 and FA-LL-37 against urogenital microflora. Microbes Infect 2:773–777
Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS, Hiemstra PS, Borregaard N (2001) Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 97:3951–3959
Stahle-Backdahl M, Heilborn J, Carlsson A, Bogentoft C (2008) Use of the cathelicidin LL-37 and derivatives thereof for wound healing, US Patent 7452864
Tabor S, Richardson CC (1984) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078
Tanaka D, Miyasaki KT, Lehrer RI (2000) Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp. to the bacterial action of LL-37: a cathelicidin found in human leucocytes and epithelium. Oral Microbiol Immunol 15:226–231
Tjabringa GS, Ninaber DK, Drijfhout JW, Rabe KF, Hiemstra PS (2006) Human cathelicidin LL-37 is a chemoattractant for eosinophils and neutrophils that acts via formyl-peptide receptors. Int Arch Allergy Immunol 140:103–112
Turner J, Cho Y, Dinh NN, Waring AJ (1998) Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42:2206–2214
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119
Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75:39–48
Zanetti M (2005) The role of cathelicidins in the innate host defenses of mammals. Curr Issues Mol Biol 7:179–196
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Krahulec, J., Hyršová, M., Pepeliaev, S. et al. High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli . Appl Microbiol Biotechnol 88, 167–175 (2010). https://doi.org/10.1007/s00253-010-2736-7
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DOI: https://doi.org/10.1007/s00253-010-2736-7