Abstract
The antilisterial class IIa bacteriocins, plantaricin 423 and mundticin ST4SA, have previously been purified from the cell-free supernatants of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, respectively. Here, we present the fusions of mature plantaricin 423 and mundticin ST4SA to His-tagged green fluorescent protein (GFP) for respective heterologous expression in Escherichia coli. Fusion of plantaricin 423 and mundticin ST4SA to His-tagged GFP produced the fusion proteins GFP-PlaX and GFP-MunX, respectively. Both fusion proteins were autofluorescent, circumvented inclusion body formation and lowered the toxicity of class IIa bacteriocins during heterologous expression. Not only did GFP-class IIa fusion stabilize heterologous expression and boost yields, the fluorescent intensity of GFP-PlaX and GFP-MunX could be monitored quantitatively and qualitatively throughout expression and purification. This robust fluorometric property allowed rapid optimization of conditions for expression and bacteriocin liberation from GFP via the incorporated WELQut protease cleavage sequence. Incubation temperature and IPTG concentration had a significant effect on bacteriocin yield, and was optimal at 18°C and 0.1–0.2 mM, respectively. GFP-MunX was approximately produced at a yield of 153.30 mg/L culture which resulted in 12.4 mg/L active mundticin ST4SA after liberation and HPLC purification. While GFP-PlaX was produced at a yield of 121.29 mg/L culture, evidence suggests heterologous expression resulted in conformation isomers of WELQut liberated plantaricin 423.
Highlights
Peptides are ubiquitous in physiological systems where they fulfill functions which are fundamental to life
In order to take advantage of green fluorescent protein (GFP) as a fusion partner, the genes encoding mature plantaricin 423 and mundticin ST4SA were fused to the C-terminus of His-tagged GFP in the pRSF-GFP-PlaX and pRSF-GFP-MunX plasmid constructs, respectively (Supplementary Figures S5a,b)
In an attempt to increase the usefulness of a bacteriocin fusion partner, we evaluated GFP as a fluorescent fusion partner for the heterologous expression of plantaricin 423 and mundticin ST4SA
Summary
Peptides are ubiquitous in physiological systems where they fulfill functions which are fundamental to life. Peptides function as hormones, neurotransmitters, growth factors, ion channel ligands or antimicrobials which makes them an attractive therapeutic resource (Otvos and Wade, 2014; Recio et al, 2017; Pfalzgraff et al, 2018; Kumar, 2019). Peptides comprised of natural amino acids are not always ideal drug candidates due to their low oral bioavailability, poor stability and short plasma half-life (Otvos and Wade, 2014; Fosgerau and Hoffmann, 2015). Using AMPs to combat resistance is more a task of elucidating peptides which provide a specific function within a specific environment and understanding the mechanisms which drive their activity. Any technique to improve the yield or spectrum of producible peptides is highly valuable, especially for uncharacterized AMPs identified through genome mining
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