title: Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system.
abstract: In this work, the intein fusion approach was used for expression and purification of cathelicidin-like peptide SMAP-29 from Escherichia coli cultures. To overcome the high toxicity of the antimicrobial peptide against host cells, both C- and N-terminal fusions with Sce VMA intein were evaluated. The fusion of SMAP-29 with the N-terminus of intein had a dramatic lethal effect. In contrast, chimeric constructs harboring SMAP-29 linked to the C-terminus of intein displayed no significant inhibition of bacterial growth. Expression of intein-SMAP fusion protein was then induced in ER2566 E. coli strain by IPTG addition and different experimental conditions were tested in order to optimize the recovery of the soluble protein complex. Peptide purification was carried out by affinity chromatography: the chitin binding domain linked to intein was used to immobilize the chimeric protein on a chitin column and intein-mediated splicing of target peptide was obtained by thiol addition. Microbroth dilution assay showed that recombinant SMAP-29 displayed a high, dose-dependent bactericidal activity. These data demonstrate that the fusion of SMAP-29 with C-intein was able to inactivate the antimicrobial properties of the cathelicidin peptide allowing the expression of fusion protein in the host cell. The intein-mediated purification supplied an effective way to recover the fusion partner in its proper biologically active form.
authors.affiliation: Dipartimento di Scienze Agrarie e Ambientali, Università di Udine, via delle Scienze, 208-33100 Udine, Italy. carla.morassutti@uniud.it
authors.forename: Carla
authors.initials: C
authors.lastname: Morassutti
authors.forename: Francesca
authors.initials: F
authors.lastname: De Amicis
authors.forename: Antonella
authors.initials: A
authors.lastname: Bandiera
authors.forename: Stefano
authors.initials: S
authors.lastname: Marchetti
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D005240:Feasibility Studies; D005822:Genetic Vectors; D047668:Inteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D054804:Cathelicidins
chemical_list: D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D054804:Cathelicidins