Abstract
CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.
Highlights
In the past few decades, as multidrug-resistant pathogens spread at an alarming rate, many efforts have been made to develop new therapeutic compounds that can be used to safely treat infectious diseases [1,2,3,4]
We report on a novel hybrid peptide CLP with the combination of CM4 (1–8), LL37 (17–30) and TP5 (1–5) for its improving antibacterial activity and minimizing cytotoxic effects
The expression plasmid pET-SUMO-TEV-CLP was constructed by synthesizing the His-SUMO-TEV-CLP gene and cloning it into pET-Amp
Summary
In the past few decades, as multidrug-resistant pathogens spread at an alarming rate, many efforts have been made to develop new therapeutic compounds that can be used to safely treat infectious diseases [1,2,3,4]. Antimicrobial peptides (AMPs) are a potential alternative to antibiotic treatment [5,6]. They are amphipathic, cationic, and small polypeptides [7]. AMPs exhibit antimicrobial activity through various mechanism models, common models are “barrel-stave”, “carpet” and “toroidal-pore” [12,13,14]. These sterilization mechanisms can reduce the production of resistant bacteria. AMPs show great promise as a new type of antibacterial agent, which can kill antibiotic-resistant bacterial pathogens
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