Characterization of factors favoring the expression and purification of recombinant LL-37 from Escherichia coli

  • Ja-Young Moon
  • Dae-Ook Kang
  • Yong-Kweon Cho
  • Kwang-Hoon Kong
  • Dong-Kuk Lee
  • Ayyalusamy Ramamoorthy
Bioactive Materials Article


The only human antimicrobial peptide, LL-37, was overexpressed in soluble form using Escherichia coli-based expression system. Recombinant LL-37 production in E. Coli was optimized for use in large quantities for studying antimicrobial activity against Helicobacter pylori strains. We previously reported a method to express and purify LL-37 using Glutathione S-transferase (GST) fusion system. Herein presents method suitable for producing recombinant LL-37 from the recombinant GST-LL-37 fusion protein by recovering from both soluble protein fractions and inclusion bodies. Compared to the yield reported previously, the yield of recombinant GST-LL-37 protein was much improved to 10 mg/L of culture by affinity chromatography using GSTrap FF (1 mL) affinity chromatography column. These results suggest that the production method used in present study is useful in obtaining large quantity of recombinant LL-37 peptide. The optimized recovery protocol from inclusion bodies was highly contributable in raising yield of GST-LL-37 fusion protein. When treated with Factor Xa, GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies released an approximate 4.5-kDa protein, which was the expected size of LL-37; GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies was also confirmed by enzymatic digestion with thrombin, which produced LL-37 peptide containing six extra amino acid residues, Gly-Ile-Ile-Glu-Gly-Arg, on Nterminus of LL-37. Purified recombinant LL-37 showed nearly identical antimicrobial activities against H. Pylori strains as that of synthetic LL-37, suggesting its application to functional study with therapeutic potential on gastric pathogen.

Key words

antimicrobial peptide factor Xa glutathione S-transferase fusion protein Helicobacter pylori inclusion bodies LL-37 thrombin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Agerberth B, Gunne H, Odeberg J, Kogner P, Boman HG, and Gudmundsson GH (1995) FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc Natl Acad Sci USA 92, 195–199.Google Scholar
  2. Agerberth B, Charo J, Werr J, Olsson B, Idali F, Lindbom L, Kiessling R, Jornvall H, Wigzell H, and Gudmundsson GH (2000) The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 96, 3086–3093.Google Scholar
  3. Bals R, Wang X, Zasloff M, and Wilson JM (1998) The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA 95, 9541–9546.Google Scholar
  4. Ciornei CD, Sigurdardottir T, Schmidtchen A, and Bodelsson M (2005) Antimicrobial and chemoattractant activity, lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37. Antimicrob Agents Chemother 49, 2845–2850.Google Scholar
  5. Ciornei CD, Egesten A, and Bodelsson M (2003) Effects of human cathelicidin antimicrobial peptide LL-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro. Acta Anaesthesiol Scand 47, 213–220.Google Scholar
  6. Di Nardo A, Vitiello A, and Gallo RL (2003) Cutting edge: Mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide. J Immunol 17, 2274–2278.Google Scholar
  7. Fuchs PC, Barry AL, and Brown SD (1998) In vitro antimicrobial activity of MSI-78, a Magainin analog. Antimicrob Agents Chemother 42, 1213–1216.Google Scholar
  8. Gudmundsson GH, Agerberth B, Odeberg J, Bergman T, Olsson B, and Salcedo R (1996) The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur J Biochem 238, 325–332.Google Scholar
  9. Hase K, Murakami M, Iimura M, Cole SP, Horibe Y, Ohtake T, Obonyo M, Gallo RL, Eckmann L, and Kagnoff MF (2003) Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori. Gastroenterology 125, 1613–1625.Google Scholar
  10. Heilborn JD, Nilsson MF, Kratz G, Weber G, Sorensen O, Borregaard N, and Stahle-Backdahl M (2003) The cathelicidin anti-microbial peptide LL-37 is involved in reepithelialization of human skin wounds and is lacking in chronic ulcer epithelium. J Invest Dermatol 120, 379–389.Google Scholar
  11. Hong IP, Lee SJ, Kim YS, and Choi SG (2006) Recombinant expression of human cathelicidin (hCAP18/LL-37) in Pichia pastoris. Biotechnol Lett 29, 73–78.Google Scholar
  12. Kirikae T, Hirata M, Yamasu H, Kurikae F, Tamura H, Kayama F, Nakatsuka K, Yokochi T, and Nakano M (1998) Protective effects of a human 18-kilodalton cationic antimicrobial protein (CAP-18)-derived peptide against murine endotoxemia. Infect Immun 66, 1861–1868.Google Scholar
  13. Krahulec J, Hyrsova M, Pepeliaev S, Jilkova J, Cerny Z, and Machalkova J (2010) High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli. Appl Microbiol Biotechnol 88, 167–175.Google Scholar
  14. Larrick JW, Hirata M, Balint RF, Lee J, Zhong J, and Wright SC (1995) Human CAP 18 — A novel antimicrobial lipopolysaccharide-binding protein. Infect Immun 63, 1291–1297.Google Scholar
  15. Leszczynska K, Namiot A, Fein DE, Wen Q, Namiot Z, Savage PB, Diamond S, Janmey PA, and Bucki R (2009) Bactericidal activities of the cationic steroid CSA-13 and the cathelicidin peptide LL-37 against Helicobacter pylori in simulated gastric juice. BMC Microbiol 9, 187.Google Scholar
  16. Li Y, Li X, Li H, Lockridge O, and 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.Google Scholar
  17. McGee DJ, George AE, Trainor EA, Horton KE, Hildebrandt E, and Testerman TL (2011) Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37. Antimicrob Agents Chemother 55, 2897–2904.Google Scholar
  18. Mendez-Samperio P (2010) The human cathelicidin hCAP18/ LL-37: A multifunctional peptide involved in mycobacterial infections. Peptides 31, 1791–1798.Google Scholar
  19. Moon JY, Henzler-Wildman KA, and Ramamoorthy A (2006) Expression and purification of a recombinant LL-37 from Escherichia coli. BBA 1758, 1351–1358.Google Scholar
  20. Nicolas P and Mor A (1995) Peptide as weapons against microorganisms in the chemical defense system of vertebrates. Annu Rev Microbiol 49, 277–304. Niyonsaba F, Iwabuchi K, Someya A, Hirata M, Matsuda HGoogle Scholar
  21. Ogawa H, and Nagaoka I (2002) A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis. Immunology 106, 20–26.Google Scholar
  22. Niyonsaba F, Someya A, Hirata M, Ogawa H, and Nagaoka I (2001) Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D-2 production from mast cells. Eur J Immunol 31, 1066–1075.Google Scholar
  23. Putsep K, Carlsson G, Boman HG, and Andersson M (2002) Deficiency of antimicrobial peptides in patients with morbus Kostmann: An observation study. Lancet 360, 1144–1149.Google Scholar
  24. Scott MG, Davidson DJ, Gold MR, Bowdish D, and Hancock REW (2002) The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses. J Immunol 169, 3883–3891.Google Scholar
  25. Sorensen O, Arnljots K, Cowland JB, Bainton DF, and Borregaard N (1997) The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood 90, 2796–2803.Google Scholar
  26. Travis SM, Anderson NN, Forsyth WR, Espiritu C, Conway BD, Greenberg EP, McCray PB Jr., Lehrer RI, Welsh MJ, and Tack BF (2000) Bactericidal activity of mammalian cathelicidin-derived peptides. Infect Immunol 68, 2748–2755.Google Scholar
  27. Turner J, Cho Y, Kinh NN, Waring AJ, and Lehrer RI (1998) Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob Agents Chemother 42, 2206–2214.Google Scholar
  28. Yang D, Chertov O, and Oppenheim JJ (2001) Participation of mammalian defensins and cathelicidins in anti-microbial immunity: Receptors and activities of human defensins and cathelicidin (LL-37). J Leukoc Biol 69, 691–697.Google Scholar
  29. Yang D, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J, Oppenheim JJ, and Chertov O (2000) LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J Exp Med 192, 1069–1074.Google Scholar
  30. Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75, 39–48Google Scholar

Copyright information

© Korean Society for Applied Biological Chemistry and Springer 2011

Authors and Affiliations

  • Ja-Young Moon
    • 1
  • Dae-Ook Kang
    • 1
  • Yong-Kweon Cho
    • 1
  • Kwang-Hoon Kong
    • 2
  • Dong-Kuk Lee
    • 3
  • Ayyalusamy Ramamoorthy
    • 4
  1. 1.Department of Biochemistry & Health SciencesChangwon National UniversityChangwon, GyungnamRepublic of Korea
  2. 2.Department of Chemistry, College of Natural SciencesChung-Ang UniversitySeoulRepublic of Korea
  3. 3.Department of Fine chemistrySeoul National University of TechnologySeoulRepublic of Korea
  4. 4.Department of Chemistry and Biophysics Research DivisionUniversity of MichiganAnn ArborUSA

Personalised recommendations