High-efficient Expression And Activity Assay Of Recombinant Soluble Human EGF And BFGF

Both Epidermal Growth Factor(EGF)and Basic Fibroblast Growth Factor(bFGF)are members of the cell Growth Factor family and have extensive biological effects.In this study,a variety of gene mutations or knockout were designed for human EGF and bFGF sequences.For example,the 4th serine in the EGF sequence was replaced by threonine,and leucine or threonine were added into this sequence.The 25th serine of bFGF was replaced by cysteine.The synthesized EGF and bFGF gene sequences were connected to the expression vectors that fused PLB1 domain or DsbA partner tags.the recombinant plasmid was transformed into E.coli competent cells.The dominant monoclonal strain was first screened in the flask shaking stage,the effects of different culture media and conditions on the density of engineering bacteria were investigated,the expression conditions of EGF and bFGF fusion proteins were optimized.Secondly,the effects of different binding buffer and elution buffer on protein separation were compared.The target protein was purified by nickel affinity chromatography and ion exchange chromatography.Finally,the biological activity of EGF and bFGF protein stock solution was determined by MTT colorimetry,the results showed that the two protein extracts could significantly promote the proliferation of NIH-3T3 cells,the titer of EGF was3.829×10~5 IU/mg,the titer of bFGF was 3.518×10~5 IU/mg.A variety of EGF and bFGF mutation sequences were constructed in the upstream design phase,the expression of target protein was increased.The fermentation and culture conditions of engineering bacteria were optimized.In the separation and purification stage,follow the principle that the fewer steps,the higher the yield,make the chromatography operation into two steps,and the purity of the product can reach more than 90%.It can meet the requirements of the following experiments,such as specificity identification and activity detection.The structure of the protein stock solution was verified by Western Blot,and according to the criterion of pharmacopoeia,a scientific,rigorous and low-cost method for biological activity detection was established,the biological activity of EGF and bFGF was detected by this method.It lays a foundation for the industrial production of EGF and bFGF.