Preparation Of The Elastin-like Polypeptides-Epidermal Growth Factor (ELPs-EGF) Fusion Protein And Its Effect On Skin Injury Repair

Epidermal growth factor（EGF）is a small peptide consisting of 53 amino acids with a molecular weight of 6.2 kDa.It has excellent acid stability and thermostability.EGF can produce good coordination effects with various enzymes in vivo,promote the mitosis and proliferation of hepatic epithelial cells,accelerate the metabolism of skin and facilitate epithelial regeneration.Many studies have shown EGF exhibited regulatory effects on reproductive system,alimentary system and nervous system.Elastin-Like Polypeptides（ELPs）are proteins with the（VPGXG）n sequence units,in which X can be any amino acid other than Pro.ELPs are developed by recombinant engineering of the elastin VPGVG motif.ELPs have good biocompatibility,low immunogenicity and can be degraded into natural amino acids in vivo.In addition,ELPs can form a hydrogel at a specific temperature at its phase transition temperature and therefore are considered to be an relative ideal drug release carrier.The structure of the natural multifunctional protein inspired the researchers to design a multifunctional fusion protein with different combinations by using genetic engineering technology.It has been reported that the reconstructed elastin fused with some protein has both ELPs activity and the protein’s activity.In order to obtain the fusion protein with the properties of ELPs and human EGF,genetic recombination technology was used to fuse ELPs with human EGF,from which foundation was laid for the research and development of novel tissue engineering materials.Results were shown as follows:First,the recursive directional ligation（RDL）method was used to obtain ELP50 with amino acid sequence[（VPGVG）₁₀VPGKG]₅,which was then fused with EGF group to construct eukaryotic expression vector pET28a-ELP50 and pET28a-ELP50-EGF,respectively.The two expression vectors were introduced into E.coli strain BL21（DE3）,and the recombinant proteins ELP50 and ELP50-EGF were expressed under the induction of Isopropyl-β-D-Thiogalactoside.Secondly,the recombinant protein ELP50 with a purity over 90%can be obtained by primary purification at alternating temp followed by inverse transition cycling（ITC）method.On the other hand,the recombinant ELP50-EGF was purified by using the nickel column affinity chromatography with a similar purity at above 90%.Thirdly,the proliferation of mouse fibroblast cell line L929 under different treatment condition indicated that ELP50-EGF did not promote cell proliferation compared to CA,ELP50 and EGF treatment at 48 h.However,at 72 h the ELP50-EGF treatment group exhibited significant proliferation stimulating effect compared with PBS（p<0.001）,CA（p<0.001）,ELP50（p<0.01）and EGF（p<0.05）treatment groups.Finally,the skin defect repair experiment of Kunming mice revealed that the skin repair of all groups were completed at 12 days post injury,and the ELP50-EGF group had improved healing effect compared with other groups,which was verified by H&E staining of tissue sections.The histological section staining showed faster epithelial repair,more even epidermis layer,and increased a large number of epithelial cells in ELP50-EGF group compared to control,CA,ELP50 and EGF groups.The statistical analysis further indicated that the thickness of repaired skin of ELP50-EGF group was significantly different from that of control,CA,ELP50 and EGF（p<0.001）treatment groups.Meanwhile,the dermis repair of ELP50-EGF group is also noticeable,with plentiful fibroblasts could be seen.Conclusion:The ELP50-EGF fusion protein exents a significant promoting effect on the proliferation of L929 cells,and accelerates skin process repair on our mouse skin model.