Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

• Published in: Molecules (Basel, Switzerland) Switzerland), 2021-11, Vol.26 (23), p.7142
• Main Authors: Cheng, Junhao, Ahmat, Marhaba, Guo, Henan, Wei, Xubiao, Zhang, Lulu, Cheng, Qiang, Zhang, Jing, Wang, Junyong, Si, Dayong, Zhang, Yueping, Zhang, Rijun
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - biosynthesis; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; Antibacterial activity; Antibiotics; Antimicrobial activity; Antimicrobial agents; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - genetics; antimicrobial peptide; Antimicrobial peptides; Antimicrobial Peptides - biosynthesis; Antimicrobial Peptides - chemistry; Antimicrobial Peptides - genetics; Antimicrobial Peptides - pharmacology; Bacteria; Bacteria - drug effects; Bacteria - pathogenicity; Bacterial diseases; Biological activity; Cathelicidins - chemistry; Cathelicidins - genetics; Cell death; Cloning; E coli; Endopeptidase K; Escherichia coli; Escherichia coli - genetics; fusion expression; Fusion protein; Gram-negative bacteria; Hemolysis - drug effects; Humans; hybrid peptide; Iodides; Morphology; Multidrug resistance; Papain; Pathogens; Pepsin; Peptides; Peptides - chemistry; Peptides - genetics; Peptides - pharmacology; Plasmids; Propidium iodide; Proteinase; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacology; Scanning electron microscopy; Sheep; Stability analysis; Thermal stability; Toxicity; Trypsin
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules26237142

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 . 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 (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.