title: Escherichia coli expression and purification of LL37 fused to a family III carbohydrate-binding module from Clostridium thermocellum.
abstract: The cathelicidin derived human peptide LL37 has a broad spectrum of antimicrobial and immunomodulatory activities. The large variety of biological activities makes LL37 a very promising candidate for clinical applications. The production of biologically active LL37 in large amounts with reduced costs can only be achieved using recombinant techniques. In this work, LL37 has been cloned to the N- and C-termini of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; both constructions (LL37-LK-CBM3 and LK-CBM3-LL37) were cloned into the pET-21a vector. A formic acid recognition site was introduced between the two modules, allowing the isolation of LL37 after chemical cleavage. The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and solubilized with Triton X-100. The purification was achieved using cellulose CF11 fibers, taking advantage of the CBM3 specific affinity for cellulose; after hydrolysis with formic acid, LL37 was further purified by reverse-phase HPLC, as confirmed by MALDI-TOF mass spectrometry. The production and purification methodology developed in this work compares advantageously to other protocols previously described, having fewer purification steps. Only the recombinant LL37 obtained from the C-terminally fused protein (LK-CBM3-LL37) showed antibacterial activity against E. coli K12, with a MIC of 180 microg/ml.
authors.affiliation: Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
authors.forename: Reinaldo
authors.initials: R
authors.lastname: Ramos
authors.forename: Lucília
authors.initials: L
authors.lastname: Domingues
authors.forename: Miguel
authors.initials: M
authors.lastname: Gama
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D048013:Acetivibrio thermocellus / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D054804:Cathelicidins
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C055213:saccharide-binding proteins; D054804:Cathelicidins