title: A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes.
abstract: BACKGROUND
  To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.
  
  
  RESULTS
  Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.
  
  
  CONCLUSIONS
  The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.
authors.affiliation: Department of Life Science and Technology, Nanyang Normal University, Wolong Road, Nanyang 473061, China. ketao1@gmail.com
authors.forename: Tao
authors.initials: T
authors.lastname: Ke
authors.forename: Su
authors.initials: S
authors.lastname: Liang
authors.forename: Jin
authors.initials: J
authors.lastname: Huang
authors.forename: Han
authors.initials: H
authors.lastname: Mao
authors.forename: Jibao
authors.initials: J
authors.lastname: Chen
authors.forename: Caihua
authors.initials: C
authors.lastname: Dong
authors.forename: Junyan
authors.initials: J
authors.lastname: Huang
authors.forename: Shengyi
authors.initials: S
authors.lastname: Liu
authors.forename: Jianxiong
authors.initials: J
authors.lastname: Kang
authors.forename: Dongqi
authors.initials: D
authors.lastname: Liu
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Ma
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D008826:Microbial Sensitivity Tests; D016982:Micrococcus luteus / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D014764:Viral Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C505229:N(pro) protein, swine fever virus