title: Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli.
abstract: Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12h induction with 0.5mM IPTG (isopropyl beta-d-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 μM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 μM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.
authors.affiliation: Group of Molecular Immunology, Faculty of Health Sciences, Quindío University, Cra 15 calle 12 norte, Armenia Quindío, Colombia. Electronic address: gymol@uniquindio.edu.co.
authors.forename: Germán Alberto
authors.initials: GA
authors.lastname: Téllez
authors.affiliation: Group of Molecular Immunology, Faculty of Health Sciences, Quindío University, Cra 15 calle 12 norte, Armenia Quindío, Colombia.
authors.forename: Jhon Carlos
authors.initials: JC
authors.lastname: Castaño-Osorio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D001483:Base Sequence; D054807:Cecropins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods; D004175:Diptera / Q000737:chemistry / Q000235:genetics*; D024901:Drug Resistance, Multiple, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects*; D004927:Escherichia coli Infections / Q000188:drug therapy; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014709:Vero Cells
chemical_list: D000900:Anti-Bacterial Agents; D054807:Cecropins; D011993:Recombinant Fusion Proteins
keywords: Antimicrobial peptide; Cecropin; Chromatography; Drug resistance; Insect; Recombinant fusion proteins