A Hybrid Peptide DEFB-TP5 Expressed in Methylotrophic Yeast Neutralizes LPS With Potent Anti-inflammatory Activities

Abstract

DEFB-TP5 is a novel auspicious health-beneficial peptide derivative from two naturally occurring peptides, β-Defensin (DEFB) and thymopentin (TP5), and shows strong anti-inflammatory activity and binds to LPS without cytotoxicity and hemolytic effect. Furthermore, the application of DEFB-TP5 peptide is inadequate by its high cost. In the current study, we developed a biocompatible mechanism for expression of the DEFB-TP5 peptide in Pichia pastoris. The transgenic strain of hybrid DEFB-TP5 peptide with a molecular weight of 6.7kDa as predictable was obtained. The recombinant DEFB-TP5 peptide was purified by Ni-NTA chromatography, estimated 30.41 mg/L was obtained from the cell culture medium with 98.2% purity. Additionally, The purified DEFB-TP5 peptide significantly (p< 0.05) diminished the release of nitric oxide (NO), TNF-α, IL-6, IL-1β in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner. This study will not only help to understand the molecular mechanism of expression that can potentially be used to develop an anti-endotoxin peptide but also to serve as the basis for the development of antimicrobial and anti-inflammatory agents as well, which also provides a potential source for the production of recombinant bioactive DEFB-TP5 at the industrial level.

Keywords: anti-inflammatory; endotoxin; expression; hybrid peptide; β-defensins.

Figure 1

Tricine-SDS-PAGE and analysis of recombinant peptide, (A) Tricine-SDS-PAGE of the cell culture media from P.pastoris expressing secreted DEFB-TP5. Lane M, mass weight markers; Lane C, control (blank PpICZαA and X-33 strain); Lane 1 to 7 (supernatant X33/PpICZαA-DEFB-TP5) peptide expression after methanol (12 to 144 h) induction and arrow in the lane indicated 6.7 kDa peptide (B) Tricine-SDS-PAGE of Purified secreted recombinant hybrid peptide DEFB-TP5. Lane M, mass weight markers; Lane C, control (blank PpICZαA and X-33 strain); Lane 1-6 purified X33/DEFB-TP5 extract with different concentrations of imidazole and arrow in the lane indicated 6.7 kDa (400 and 500mm imidazole) polypeptide. (C) The antimicrobial activity of recombinant DEFB-TP5 against E.coli C 84002, A: 100 U Ampicillin sodium, B: recombinant hybrid DEFB-TP5 peptide (concentration 5mg/L), C: The negative control, sodium phosphate buffer (PBS).

Figure 2

LPS neutralization, cytotoxicity, and hemolytic activity of parental and recombinant DEFB-TP5 peptide. (A) Endotoxin binding by means of an endotoxin quantitation kit. Mean values presented; n = 3 ± SD (*p < 0.05, **p < 0.01 and ***p < 0.001 showed comparison of LPS vs. DEFB-TP5. Whereas, ^#p < 0.05 showed significant difference compared with parental TP5 peptide). (B) hybrid peptide reduced LDH in the supernatant of LPS-stimulated mouse RAW264.7 macrophages. Data represented as mean ± standard deviation (SD). While, *p < 0.05 and **p < 0.01 vs. LPS and ^#p < 0.05 indicates significant difference compared with parental TP5 peptide. (C) Hemolytic effect of DEFB-TP5 in contradiction of mouse RBCs. The data resemble the mean values of 3-independent experiments and the (% age) of hemolysis ± standard deviation (***p < 0.001 vs. Triton X-100) While, ^#p < 0.05 showed a comparison with TP5.

Figure 3

Effect of TP5 and recombinant DEFB-TP5 peptide on LPS-infected inflammatory response in mouse RAW264.7 macrophages. (A) Nitric oxide (NO) production, (B) level of Tumor necrosis factor-a, (C) Interleukin-6, and (D) Interleukin-1b. Standards are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, showed comparsion with LPS. While, #p < 0.05 and ##p < 0.01 indicates significant difference compared with parental TP5 peptide.