Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization

• Published in: Microorganisms (Basel) Vol. 11; no. 1; p. 131
• Main Authors: Cheng, Junhao, Ahmad, Baseer, Raza, Muhammad Asif, Guo, Henan, Ahmat, Marhaba, Wei, Xubiao, Zhang, Lulu, Li, Zhongxuan, Cheng, Qiang, Zhang, Jing, Wang, Junyong, Si, Dayong, Zhang, Yueping, Zhang, Rijun
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 04.01.2023; MDPI
• Subjects: Affinity chromatography; Amino acids; Antibiotics; Apoptosis; Bacteria; Bacterial infections; Chemical compounds; Cloning; Cloning vectors; Cytokines; Cytotoxicity; E coli; endotoxin; Endotoxins; Enzymes; Gel electrophoresis; Genomes; Glycerol; hybrid peptide; IL-1β; immunomodulatory; Inflammation; Lipopolysaccharides; Macrophages; Mass spectrometry; Mass spectroscopy; Neutralization; Nitric oxide; Peptides; Pharmacology; Plasmids; Potassium; Sepsis; Sodium lauryl sulfate; Tumor necrosis factor-α; Yeast; Yeasts; Beijing China; United States > US; China; hybrid peptide; apoptosis; inflammation; cytotoxicity; endotoxin; immunomodulatory
• ISSN: 2076-2607; 2076-2607
• DOI: 10.3390/microorganisms11010131

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.