title: A high-throughput expression screening platform to optimize the production of antimicrobial peptides.
abstract: BACKGROUND
  Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library.
  
  
  RESULTS
  The combinatorial library is based on the Golden Gate cloning system. In each reaction, six donor plasmids (each containing one component: a promoter, fusion partner 1, fusion partner 2, protease cleavage site, gene of interest, or transcriptional terminator) were combined with one acceptor plasmid to yield the final expression construct. As a proof of concept, screening was carried out in Escherichia coli and Pichia pastoris to study the expression of three different model AMPs with challenging characteristics, such as host toxicity or multiple disulfide bonds. The corresponding genes were successfully cloned in 27 E. coli and 18 P. pastoris expression plasmids, each in a one-step Golden Gate reaction. After transformation, small-scale expression screening in microtiter plates was followed by AMP quantification using a His6tag-specific ELISA. Depending on the plasmid features and the expression host, the protein yields differed by more than an order of magnitude. This allowed the identification of high producers suitable for larger-scale protein expression.
  
  
  CONCLUSIONS
  The optimization of recombinant protein production is best achieved from first principles by initially optimizing the genetic construct. The unrestricted combination of multiple plasmid features yields a comprehensive library of expression strains that can be screened for optimal productivity. The availability of such a platform could benefit all laboratories working in the field of recombinant protein expression.
authors.affiliation: University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Schreiber
authors.affiliation: University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany.
authors.forename: Hagen
authors.initials: H
authors.lastname: Müller
authors.affiliation: University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Birrenbach
authors.affiliation: University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Klein
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group Bioresources, Giessen, Germany.
authors.forename: Doreen
authors.initials: D
authors.lastname: Heerd
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group Bioresources, Giessen, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Weidner
authors.affiliation: University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany. denise.salzig@lse.thm.de.
authors.forename: Denise
authors.initials: D
authors.lastname: Salzig
authors.affiliation: University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany. peter.czermak@lse.thm.de.|Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group Bioresources, Giessen, Germany. peter.czermak@lse.thm.de.|Faculty of Biology and Chemistry, Justus Liebig University Giessen, Giessen, Germany. peter.czermak@lse.thm.de.|Department of Chemical Engineering, Kansas State University, Manhattan, USA. peter.czermak@lse.thm.de.
authors.forename: Peter
authors.identifier: http://orcid.org/0000-0001-7485-500X
authors.initials: P
authors.lastname: Czermak
mesh_terms: D000890:Anti-Infective Agents / Q000302:isolation & purification*; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D011994:Recombinant Proteins
keywords: Antimicrobial peptides; Combinatorial library; E. coli; Expression screening; Expression strain; Fusion protein; Golden Gate cloning; P. pastoris; Promoter; Recombinant protein production