Expression, Purification And Bioactivity Of The Human Elastin-like Polypeptide Fusion Protein Combining Basic Fibroblast Growth Factor

Biomaterials played an important role in the research and practice of tissue engineering and regenerative medicine. It was an important target of the biomaterials research to develop a new type of material which could not only be the support for seed cells but also be able to promote cell proliferation. Elastin-like polypeptide was a kind of gene engineering of synthetic polypeptides which was based on the amino acid sequence. It was a widely used in tissue engineering and controlled sustained-release drug areas due to its many characteristics such as self-assembly and temperature sensitivity. To make fusion protein combining ELP and growth factor which had specific bioactivity was meaningful for study and practice.Objective:To design and synthesize the gene sequence of hELPs-bFGF, and to construct the prokaryotic expression vector of hELPs-bFGF; to explore prokaryotic expression conditions of hELPs-bFGF and establishment the separation and purification methods of this protein; to evaluate the biological properties of this protein to provide a basis for further research.Methods and Results:①The synthetic elastin-like polypeptide sequences were amplified from [(VAPGVG)3S]4to [(VAPGVG)3S]32using recursive directional ligation. Then, the gene segment was mixed and linked to synthetic bFGF gene segment and pET28a expression vector segment obtained by double digestion according to molecular molar ratio of2:2:1to acquire the prokaryotic expression vector pET28a-hELPs-bFGF.②Single factor experiments were designed to explore the impact of various factors under different conditions on the target protein expression. Based on the above result, an orthogonal test was operated to get the optimal expression conditions for the fermentation of proteins. The result indicated that the optimal conditions for the expression of the target protein were pH=7.0, OD600=0.8(induced timing), and6h(induced length of time). The relative expression level of the target protein in fermentation product was about27.4%.③The target protein was identified by Western-Blot. His-tag and bFGF western-blot results were both positive. The result demonstrated that the protein with molecular weight of70.4kDa on the PVDF film was the hELPs-bFGF. The target protein was purified by His-tag affinity chromatography after the thalli had been smashed and washed.④The lyophilized products of hELPs-bFGF was applied on the mice skin defects. Experimental results showed that the fusion protein can promote skin defects regeneration in mice significantly.Conclusion:The result indicated that the hELPs-bFGF protein could be synthesized successfully by genetic engineering technology. The hELPs-bFGF protein could be expressed correctly in the engineering bacteria. Optimization of expression conditions contributed to improving the expression of the target protein. The lyophilized protein could promote skin defects regeneration in mice significantly, which added a new selection of biological material for clinical use.