A high-throughput expression screening platform to optimize the production of antimicrobial peptides

Microbial Cell Factories

Research

Tobias Weidner¹  Doreen Heerd¹  Moritz Klein²  Christine Schreiber²  Oliver Birrenbach²  Hagen Müller²  Denise Salzig²  Peter Czermak³ 

[1] Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group Bioresources, Giessen, Germany;University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany;University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390, Giessen, Germany;Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group Bioresources, Giessen, Germany;Faculty of Biology and Chemistry, Justus Liebig University Giessen, Giessen, Germany;Department of Chemical Engineering, Kansas State University, Manhattan, USA;

关键词: Antimicrobial peptides;    Expression screening;    Recombinant protein production;    Golden Gate cloning;    Combinatorial library;    E. coli;    P. pastoris;    Expression strain;    Promoter;    Fusion protein;   

DOI  :  10.1186/s12934-017-0637-5

 received in 2016-11-30, accepted in 2017-01-21,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundAntimicrobial 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.ResultsThe 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 His6 tag-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.ConclusionsThe 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.

【 授权许可】

CC BY   
© The Author(s) 2017