Abstract
BackgroundDespite the growing demand for antimicrobial peptides (AMPs) for clinical use as an alternative approach against antibiotic-resistant bacteria, the manufacture of AMPs relies on expensive, small-scale chemical methods. The small ubiquitin-related modifier (SUMO) tag is industrially practical for increasing the yield of recombinant proteins by increasing solubility and preventing degradation in expression systems.ResultsA new vector system, pKSEC1, was designed to produce AMPs, which can work in prokaryotic systems such as Escherichia coli and plant chloroplasts. 6xHis was tagged to SUMO for purification of SUMO-fused AMPs. Abaecin, a 34-aa-long antimicrobial peptide from honeybees, was expressed in a fusion form to 6xHis-SUMO in a new vector system to evaluate the prokaryotic expression platform of the antimicrobial peptides. The fusion sequences were codon-optimized in three different combinations and expressed in E. coli. The combination of the native SUMO sequence with codon-optimized abaecin showed the highest expression level among the three combinations, and most of the expressed fusion proteins were detected in soluble fractions. Cleavage of the SUMO tag by sumoase produced a 29-aa-long abaecin derivative with a C-terminal deletion. However, this abaecin derivative still retained the binding sequence for its target protein, DnaK. Antibacterial activity of the 29-aa long abaecin was tested against Bacillus subtilis alone or in combination with cecropin B. The combined treatment of the abaecin derivative and cecropin B showed bacteriolytic activity 2 to 3 times greater than that of abaecin alone.ConclusionsUsing a SUMO-tag with an appropriate codon-optimization strategy could be an approach for the production of antimicrobial peptides in E.coli without affecting the viability of the host cell.
Highlights
Despite the growing demand for antimicrobial peptides (AMPs) for clinical use as an alternative approach against antibiotic-resistant bacteria, the manufacture of Antimicrobial peptide (AMP) relies on expensive, small-scale chemical methods
As an alternative approach to prevent the spread of bacteria which are resistant to antibiotics, antimicrobial peptides (AMPs) have been extensively studied since they have a broad spectrum of anti-infective activity against pathogenic bacteria with relatively low minimal inhibitory concentrations and with a property less capable of incurring resistance than conventional antibiotics, due to their nonspecific interaction with bacterial membranes, and their ability to work on multiple targets
We describe a new expression platform for the efficient produciton of AMPs, which can be potentially operable in both bacteria and plant chloroplast, and its use in an expression platform such as E.coli was first tested in this study
Summary
Despite the growing demand for antimicrobial peptides (AMPs) for clinical use as an alternative approach against antibiotic-resistant bacteria, the manufacture of AMPs relies on expensive, small-scale chemical methods. The overuse of antibiotics for the last several decades and the prevalence of antibiotic-resistant bacterial infections present a threat to global health. There is a potential that over-use or abuse of a new antibiotic will give rise to more antibiotic-resistance bacteria. As an alternative approach to prevent the spread of bacteria which are resistant to antibiotics, antimicrobial peptides (AMPs) have been extensively studied since they have a broad spectrum of anti-infective activity against pathogenic bacteria with relatively low minimal inhibitory concentrations and with a property less capable of incurring resistance than conventional antibiotics, due to their nonspecific interaction with bacterial membranes, and their ability to work on multiple targets. In addition to the direct antimicrobial activities, many AMPs are associated with immunomodulatory properties as seen in host-defense peptides [3, 4]
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