title: Prokaryotic expression and mechanism of action of α-helical antimicrobial peptide A20L using fusion tags.
abstract: BACKGROUND
  Antimicrobial peptides have become important candidates as new antibiotics against resistant bacterial strains. However, the major industrial manufacture of antimicrobial peptides is chemical synthesis with high costs and in relatively small scale. The Ub-tag and SUMO-tag are useful for increasing the yield of enzymes and other proteins in expression system. In this study, antimicrobial peptide A20L (KWKSFLKTFKSAKKTVLHTLLKAISS), a derivative of V13K in the previous study is used as a template to be expressed in different Ub-tag and human SUMO tag systems to compare the prokaryotic expression approaches of antimicrobial peptide. The antibacterial mechanism of action and membrane specificity of A20L was further studied.
  
  
  METHODS
  We fused the Ub and SUMO1/2/3/4 with A20L to construct expression plasmids. Ub-A20L and SUMO1/2/34 gene sequences were inserted into the pHUE plasmids and pET-28b+ plasmids, respectively, to construct pHUE-A20L plasmids and pET-28b+-SUMO1/2/3/4-A20L plasmids. These plasmids were transformed into E. coli Rosetta (DE3) and induced with IPTG to express Ub-A20L and SUMO1/2/3/4 fusion proteins. The recombinant proteins were found in the soluble fraction after being over expressed in E. coli Rosetta (DE3). Antibacterial and hemolytic activities and membrane permeabilization ability of A20L were determined. Peptide structure was also studied by circular dichroism experiments.
  
  
  RESULTS
  A20L (KWKSFLKTFKSAKKTVLHTLLKAISS) was successfully expressed by fusion with an ubiquitin tag (Ub-tag) and human SUMO tags (SUMO1/2/3/4-tags). A20L exhibited antimicrobial activity against various Gram-negative and Gram-positive bacteria. Based on the hemolytic activity against human red blood cells, A20L showed good specificity against bacteria. The circular dichroism experiments illustrated that A20L was transferred into an α-helical structure in the presence of hydrophobic environment. The antibacterial mechanism of action and membrane specificity of A20L was further studied using membrane permeabilization experiments and tryptophan fluorescence and quenching experiments in liposomes.
  
  
  CONCLUSIONS
  The Ub-tag and human SUMO-tags represent good alternatives to chemical synthesis for the industrial production of antimicrobial peptides with low costs and high yields. The antibacterial mechanism of action of A20L was proved as membrane disruption. A20L showed stronger specificity on liposomes mimicking bacterial membrane than those mimicking eukaryotic cell membrane, which is consistent with the biological activity studies.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 Qianjin St., Changchun, Jilin, 130012, P. R. China. yitonghui@126.com.|School of Life Sciences, Jilin University, Changchun, China. yitonghui@126.com.
authors.forename: Tonghui
authors.initials: T
authors.lastname: Yi
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 Qianjin St., Changchun, Jilin, 130012, P. R. China. sunshiyu1988@aliyun.com.|School of Life Sciences, Jilin University, Changchun, China. sunshiyu1988@aliyun.com.
authors.forename: Shiyu
authors.initials: S
authors.lastname: Sun
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 Qianjin St., Changchun, Jilin, 130012, P. R. China. huangyibing@jlu.edu.cn.|National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun, China. huangyibing@jlu.edu.cn.|School of Life Sciences, Jilin University, Changchun, China. huangyibing@jlu.edu.cn.
authors.forename: Yibing
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2699 Qianjin St., Changchun, Jilin, 130012, P. R. China. chen_yuxin@jlu.edu.cn.|National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun, China. chen_yuxin@jlu.edu.cn.|School of Life Sciences, Jilin University, Changchun, China. chen_yuxin@jlu.edu.cn.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000008:administration & dosage; D023181:Antimicrobial Cationic Peptides / Q000008:administration & dosage / Q000096:biosynthesis* / Q000737:chemistry; D002462:Cell Membrane / Q000187:drug effects; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008081:Liposomes / Q000737:chemistry; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D025842:SUMO-1 Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D008081:Liposomes; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D025801:Ubiquitin