The Recombinant Expression Of Cell-Penetrating Peptide Pep-1and Human Epidemal Gowth Factor In Escherichia Coli

Epidermal growth factor (EGF) is a 6.2 kDa single-chain small polypeptide that has 53 amino acids and is secreted from various tissues. EGF is widely used in clinical for its effect in curing burns, ulceration, corneal injury and all kinds of trauma as a medicine since its function in promoting cell proliferation and epidermal growth. EGF plays an important role in skin whitening, anti-wrinkle and anti-aging as a cosmetic additive because of its function in promoting normal skin cell metabolism. Therefore, EGF has huge potential application value and broad market prospects both as medicines and cosmetics. However, studies have shown that the transmembrane efficiency of EGF is very low. The cumulative transdermal rate is only 0.993% with 0.5 μg-g-1 EGF treatment of the skin after 12 h. This indicates that most of EGF keeps staying on the cell surface and the transdermal absorption effect is very weak.Firstly, we got EGF gene from the cDNA of human breast cancer cells MDA-MB-231 using PCR technology. And then the DNA pieces of EGF and Pep-1, a CPP that have been previously shown to be powerful transport vector tools for the intracellular delivery of a variety of cargoes through the cell membrane, was spliced together with overlapping PCR technology named P-EGF. Then the EGF and P-EGF were respectively inserted into the carrier pGEX-6P-3 to build fusion expression plasmid fused with GST tag and expressed in E. coli. The most of sutible for these two kinds of protein expression was the strain of E. coli BL21-TrixB(DE3) and the best soluble fermentation medium and fermentation conditions were LB culture medium, temperature for 20℃, speed with 200 rpm, IPTG concentration with 0.4 mmol/l,time with 10 h for the protein expression of EGF, and M9 medium containing 1% glucose, temperature for 20 ℃, speed with 200 rpm, IPTG concentration with 0.2 mmol/l,time with 8 h for the another protein expression of P-EGF after optimization of expression conditions. The fusion proteins were digested at 4℃ overnight with TEV enzymes to remove the GST tag after preliminary isolation and purification with affinity chromatography technology and then we got target proteins after further sepatation and purification using ion exchange chromatography. Lastly, the biological activities and transmembrane ability of recombinant proteins were determined using MTT assay, scratch assay and immunity fluorescence assay after Western blot verification. The results we got in this study showed that the recombinant protein could promote the fibroblast cell COS-7 proliferation and migration and the fusion of Pep-1 could markedly increase the transmembrane ability of EGF, while it did not interfere the growth-stimiulating and migration-promoting functions of EGF on fibroblasts.This study successfully constructed the independent and fusion with cell-penetrating peptide Pep-1 recombinant E. coli expression system for EGF. This research not only provided a new theoretical guidance for EGF applying to clinical and cosmetic better but also laid a certain foundation for its development and production.