Heterologous Expression of Recombinant Ginseng Tetradecapeptide in Saccharomyces cerevisiae and Evaluation of Its Biological Activity

Abstract

Ginseng peptides, as bioactive components of ginseng, have attracted increasing attention. In this study, a 14-amino acid ginseng peptide was selected and heterologously expressed in Saccharomyces cerevisiae using a multicopy tandem fusion strategy, named 7RS14α. The secondary structure of the recombinant ginseng tetradecapeptide (7RS14α) was analyzed, and a high-glucose model was established in mouse adipocytes to evaluate its biological activity. Transcriptomic profiling was further performed to elucidate its potential mechanisms. Results demonstrated that 7RS14α significantly enhanced glucose uptake in high-glucose model cells, likely by modulating lipid metabolism pathways and insulin signaling cascades, thereby influencing energy homeostasis in adipocytes.

Keywords: Saccharomyces cerevisiae; diabetes mellitus; ginseng peptides; multicopy tandem expression.

Figure 1

Construction, transformation, protein expression and purification of engineered strains. (A) Graphical representation of the key steps in plasmid engineering. (B) Construction and validation of engineered strains: (a) the cloning of α-factor fragment and 7RS14-Flag fragment; (b) PCR validation of recombinant plasmids; (c) colony PCR verification of BY4741-pYES2-7RS14α. (C) Rapid detection of 7RS14α expression using colloidal gold-based FLAG-tag immunoassay kit. (D) Western blot analysis of 7RS14a protein expression. (E) Purification of 7RS14a using FLAG affinity chromatography (Lane 1–2: Flow-through fraction from fermentation broth, Lane 3: wash buffer fraction, Lane 4–6: elution fractions with varying FLAG peptide concentrations (0.1mg/mL,0.3 mg/mL,0.5 mg/mL), Lane 7–8: regeneration wash buffer fraction).

Figure 2

Systematic characterization of bioactive properties in recombinant ginseng-derived polypeptides. (A) Total I on chromatogram (TIC) analysis. (B) Sequence coverage map analysis. * indicates peptide not unique. (C) Circular dichroism (CD) spectroscopic analysis. (D) Ultraviolet-visible (UV–Vis) absorption spectroscopic analysis. (E) Fourier-transform infrared (FTIR) spectroscopic analysis.

Figure 3

Effects of 7RS14α on insulin-resistant model cells. (A) Microscopic evaluation of 3T3-L1 adipocyte differentiation: comparative analysis of pre-and post-adipogenic (Day 10) induction with oil red O staining (10×, red deposits indicate intracellular lipid accumulation). (B) Effects of treatment duration and drug type on the establishment of insulin-resistant cell models (1 μmol/L Dex and 10⁻⁶ mmol/L, 10⁻⁷ mmol/L, 10⁻⁸ mmol/L Insulin). (C) The changes in cell viability of differentiated 3T3-L1 adipocytes, treated with three concentrations of insulin for 120 h (10⁻⁶ mmol/L, 10⁻⁷ mmol/L, 10⁻⁸ mmol/L). (D) The changes in cell viability of differentiated 3T3-L1 adipocytes treated with 1 μmol/L Dex for 120 h. (E) Dose-dependent effects of recombinant ginseng polypeptides on cellular viability in differentiated 3T3-L1 adipocytes. (F) The impact of 7RS14α on glucose uptake in murine adipocytes. (G) The impact of 7RS14α on triglyceride content in murine adipocytes. * p < 0.05 versus model group, ** p < 0.01 versus model group, and *** p < 0.001 versus model group, # p < 0.05 versus control group, #### p < 0.0001 versus control group.

Figure 4

Transcriptomic analysis of 7RS14α effects on insulin-resistant adipocytes (p < 0.05). (A–C): Volcano plots of differentially expressed genes (DEGs) for comparisons: M vs. M + insulin, M vs. M + 7RS14α, and M vs. M + insulin + 7RS14α. (D–F): Hierarchical clustering heatmaps based on differentially expressed genes (DEGs) for comparisons: M vs. M + insulin, M vs. M + 7RS14α, and M vs. M + insulin + 7RS14α. (G–I): Enriched pathway maps based on differentially expressed genes (DEGs) for comparisons: M vs. M + insulin, M vs. M + 7RS14α, and M vs. M + insulin + 7RS14α.