Abstract
BackgroundAlthough Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins.ResultsIn this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes.ConclusionsThe results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents.Graphical
Highlights
Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle
Construction and verification of the THHly platform The fusion construction of the THHly platform is composed of four essential elements: protein of interest (POI), Tobacco Etch Virus (TEV) protease cleavage site (T), 6*His tag (H), and Hly218 which consists of the last 218 amino acids of hemolysin A (HlyA) (Hly), that is, the POI-THHly
A predominant band was observed in SDS-PAGE after induction with Ara and IPTG (Fig. 2c), which matches the theoretical molecular weight of the HTHHly fusion protein calculated from the amino acid sequence (26.9 kDa), indicating that the fusion protein was successfully secreted at a high level
Summary
Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins. Among the numerous expression systems, E. coli has established itself as one of the most excellent, owing to its advantages of fast and easy transformation with exogenous DNA, low-cost medium, and unparalleled growth rate which boosts rapid protein expression in a short fermentation period (Hayat et al 2018). Given the considerable advantage of one-step secretion compared to other widely applied secretion signal peptides, such as Sec B, a great deal of efforts have been made to establish T1SS for recombinant protein production, especially the use of hemolysin A (HlyA) T1SS (Thomas et al 2014a, b; Ruano-Gallego et al 2019; Khosa et al 2017)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
