title: High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli.
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 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-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.
authors.affiliation: CPN spol. s r.o., Dolní Dobrouc 401, Czech Republic. jnkrahulec@yahoo.com
authors.forename: Ján
authors.initials: J
authors.lastname: Krahulec
authors.forename: Marcela
authors.initials: M
authors.lastname: Hyrsová
authors.forename: Stanislav
authors.initials: S
authors.lastname: Pepeliaev
authors.forename: Jana
authors.initials: J
authors.lastname: Jílková
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Cerný
authors.forename: Jana
authors.initials: J
authors.lastname: Machálková
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D017123:Bacteriophage T7 / Q000235:genetics; D002176:Candida albicans / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics; D013293:Enterococcus faecalis / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008826:Microbial Sensitivity Tests; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000187:drug effects; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D054804:Cathelicidins
chemical_list: D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D054804:Cathelicidins