Cystatin-mediated enhancement of human epidermal growth factor bioproduction in plants

Abstract

Human epidermal growth factor (EGF) fused with the protease inhibitor SlCYS8 and produced in plants showed enhanced stability, yield, and biological activity. This fusion strategy reduced degradation and outperformed commercial EGF in cell-based assays, demonstrating a promising approach for producing stable, functional proteins in plant systems for pharmaceutical applications.

Figure 1

(Solanum lycopersicum) Cystatin 8 (SlCYS8) fusion enhances stability and activity of epidermal growth factor (EGF) produced in a plant‐based system (A) Stability comparison of EGF, green fluorescent protein (GFP), and fibroblast growth factor 1 (FGF) proteins targeted to the endoplasmic reticulum (ER). Total soluble proteins were extracted from Nicotiana benthamiana leaves expressing each construct and treated with the protein translation inhibitor cycloheximide (CHX). Relative protein levels were measured at the indicated time points following CHX addition. (B) Structural diagram of human EGF illustrating the six cysteine residues forming three disulfide bonds. (C) Influence of the proteasome inhibitor MG132 or the cysteine protease inhibitor E‐64 on EGF degradation rate. Protein stability assays were performed as described in (A), with indicated concentrations of MG132 or E‐64 added along with CHX. Mock samples were prepared by extracting proteins with total soluble buffer lacking MG132 or E‐64. (D) Co‐expression of SlCYS8, but not GFP, stabilizes EGF protein levels. Values represent mean ± SE from three biological replicates. Statistical significance was determined using two‐tailed Student's t‐test: ***P < 0.005. (E) Immunoblot analysis comparing accumulation of EGF protein expressed alone, fused with SlCYS8 (SlCYS8–EGF), or fused with a mutated variant SlCYS8^QP (SlCYS8^QP–EGF) in N. benthamiana leaves. Values represent mean ± SE from three biological replicates. Statistical significance was determined using two‐tailed Student's t‐test: ***P < 0.005. (F) Immunoblot analysis of EGF and FGF proteins expressed either alone or fused to SlCYS8 in N. benthamiana. (G) Schematic overview of the Engineered SlCYS8–EGF protein purification procedure. (H) Coomassie Brilliant Blue‐stained sodium dodecyl sulfate – polyacrylamide gel electrophoresis showing protein abundance at each purification step illustrated in (G). (I) Immunoblot analysis confirming purified EGF. Commercial EGF (Comm EGF) and plant‐produced EGF (P EGF) were detected using an anti‐EGF antibody. (J) Signal transduction assays using P EGF. Levels of phosphorylated EGF receptor (p‐EGFR), ERK1/2 (p‐ERK1/2), and AKT (p‐AKT) in HaCaT cells treated with EGF were assessed by immunoblotting with the corresponding phospho‐specific antibodies. (K) Wound healing assay evaluating the biological activity of P EGF. HaCaT cells were treated with equal concentrations of P EGF, Comm EGF, or P EGF fused to SlCYS8 (P CYS8–EGF) after mechanical wounding. Phosphate‐buffered saline served as a negative control. Wound closure was quantified from measurements taken across 20 independent fields. Values represent mean ± SD from the 20 fields analyzed. Statistical significance was determined using two‐tailed Student's t‐test: *P < 0.05. Each scale bar represents 500 μm.