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

Content Provider	Springer Nature Link
Author	Krahulec, Ján Hyršová, Marcela Pepeliaev, Stanislav Jílková, Jana Černý, Zbyněk Machálková, Jana
Copyright Year	2010
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.
Starting Page	167
Ending Page	175
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	88
Issue Number	1
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2010-07-13
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Human Recombinant Antimicrobial peptide Cathelicidin Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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