Applied Microbiology and Biotechnology

, Volume 88, Issue 1, pp 167–175

High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli

  • Ján Krahulec
  • Marcela Hyršová
  • Stanislav Pepeliaev
  • Jana Jílková
  • Zbyněk Černý
  • Jana Machálková
Biotechnologically Relevant Enzymes and Proteins

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.

Keywords

Human Recombinant Antimicrobial peptide Cathelicidin 

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Ján Krahulec
    • 1
    • 2
  • Marcela Hyršová
    • 1
  • Stanislav Pepeliaev
    • 1
  • Jana Jílková
    • 1
  • Zbyněk Černý
    • 1
  • Jana Machálková
    • 1
  1. 1.Dolní Dobrouč 401Czech Republic
  2. 2.Faculty of Natural Sciences, Department of Molecular BiologyComenius University in BratislavaBratislava 4Slovak Republic

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