title: Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag.
abstract: An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti-infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa-MAP 2, a polyalanine AMP. Pa-MAP 2 was fused to an ELP-intein tag where the ELP (Elastin-like polypeptide) was used to promote aggregation and fast and cost-effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa-MAP 2 fused to the N-termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa-MAP 2 showed a MIC of 25μM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa-MAP-2 can be produced in both rich and defined media at yields 50-fold higher than reported for other AMPs produced by the ELP-intein system, and in comparable yields to expression systems with protease or chemical cleavage.
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, 70790-160, Brazil; Programa de Pós-Graduação em Patologia Molecular, Universidade de Brasília, CEP 70.790-900, Brazil. Electronic address: amarods@gmail.com.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Sousa
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, 70790-160, Brazil.
authors.forename: Kelly C L
authors.initials: KCL
authors.lastname: Mulder
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, 70790-160, Brazil.
authors.forename: Kethly S
authors.initials: KS
authors.lastname: Nobre
authors.affiliation: Grupo de Engenharia Metabólica Aplicada a Bioprocessos, Instituto de Ciências Biológicas, Universidade de Brasília, CEP 70.790-900, Brazil.
authors.forename: Nádia S
authors.initials: NS
authors.lastname: Parachin
authors.affiliation: Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, 70790-160, Brazil; Programa de Pós-Graduação em Patologia Molecular, Universidade de Brasília, CEP 70.790-900, Brazil; S-Inova Biotech, Programa de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, CEP 79.117-900, Brazil.
authors.forename: Octávio L
authors.initials: OL
authors.lastname: Franco
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000191:economics / Q000302:isolation & purification; D004549:Elastin / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D016680:Genome, Bacterial; D047668:Inteins; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000191:economics / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins; C019529:polyalanine; D004549:Elastin
keywords: Antimicrobial peptide; Batch production; ELP; Intein