Abstract
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.
Highlights
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
Toxic peptides can be produced in E. coli by genetic masking using a fusion protein, e.g. thioredoxin fusions disrupt the bactericidal activity of gibberellin stimulated-like 1 (GSL1) [7]
We investigated the expression of three model recombinant AMPs with different characteristics: (1) insect metalloproteinase inhibitor (IMPI) (7.7 kDa; with five disulfide bonds) [8]; (2) BR021 (8.4 kDa; toxic towards E. coli) [4]; and (3) an antifungal peptide (AFP) from Lucilia sericata (8.2 kDa; without disulfide bonds and nontoxic to our hosts) [25]
Summary
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. Antimicrobial peptides (AMPs) form part of the innate immune system in most living organisms, and they act against a spectrum of pathogens including Grampositive and Gram-negative bacteria, viruses and fungi. These small and often cationic peptides are . SpStrongylocin 1 and 2 could not be expressed in E. coli BL21 (DE3), but were successfully produced in OverExpress C43 (DE3) [2] The drawback of these strains is that their growth and production rates are often lower than in their non-engineered counterparts
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