Fusion with pep-1, a cell-penetrating peptide, enhances the transmembrane ability of human epidermal growth factor

Figures & data

Fig. 1. Optimization of the fermentation conditions for the soluble expression of recombinant rhEGF and P-EGF.

Notes: (A) Effects of different culture mediums on the yield of soluble products; (B) Effects of different induction temperature on the yield of soluble products; (C) Effects of different rotational speed on the yield of soluble products; (D) Effects of different IPTG concentrations on the yield of soluble products; (E) Effects of different induction times on the yield of soluble products.

Fig. 2. Purification and identification of the recombinant proteins.

Notes: (A) SDS–PAGE analysis of the purification of protein rhEGF. Lane M, protein marker; (1) commercial hEGF agent (EGF); (2) purified rhEGF after glutathione affinity chromatography, TEV digestion and ion exchange chromatography; (3) purified GST-fused rhEGF after glutathione affinity chromatography; (4) proteins released from GST-fused rhEGF after TEV protease digestion. (B) SDS–PAGE analysis of the purification of GST-fused P-EGF. Lane M, protein marker; 1, the total expression products of bacteria transformed with pGEX-P-EGF; (2) purified GST-fused P-EGF after glutathione affinity chromatography. (C) SDS–PAGE analysis of the purification of P-EGF. Lane M, protein marker; (1) proteins released from GST-fused P-EGF after TEV protease digestion; (2) purified P-EGF after ion exchange chromatography. (D) Western blot analysis of the purified rhEGF and P-EGF. Lane 1, hEGF; (2) P-EGF.

Fig. 3. Effects of rhEGF and P-EGF on the migration and proliferation of COS-7 fibroblast cells.

Notes: (A) Effects of the recombinant proteins on the migration of COS-7 cells. Confluent cells cultured in 24-well plates were scratched using sterile pipette tips, and then treated with 1 ng/mL rhEGF, P-EGF or commercial hEGF agent (EGF) for 48 h. The space between the wound fronts was measured and the cell migration rate was calculated; (B) The fold mobility ratio of cells with different treatment; (C) Effects of the recombinant proteins on the proliferation of COS-7 cells. Cells were incubated with rhEGF, P-EGF, or commercial EGF for 72 h, and then the proliferation ability of cells was conducted by MTT assay.

Fig. 4. The immunofluorescence assay of the transmembrane ability of P-EGF.

Notes: COS-7 cells were incubated with rhEGF or P-EGF for 48 h, and then the immunofluorescence assay was performed. 4′, 6-diamidino-2-phenylindole (DAPI) staining was used to highlight the nuclei.