Difference between revisions of "Part:BBa K4965004"

Latest revision as of 12:13, 12 October 2023

We added a phoA secretion tag to the hEGF gene

Although E. coli Nissle 1917 can slowly secrete the hEGF growth factor, the secretion rate is relatively low, which is not efficient enough for acne treatment. Therefore, we decided to add a phoA secretion tag to the hEGF gene, facilitating the secretion of hEGF into the periplasmic space of E. coli. phoA is a signal peptide that guides newly synthesized proteins towards the secretion pathway. hEGF is the gene responsible for synthesizing hEGF, the human epidermal growth factor. We constructed a plasmid with fused phoA-hEGF gene, transformed it into E. coli, and validated its production and secretion using ELISA and Western Blot.

Usage and Biology

We used the old brick hEGF as the basis for fusing the phoA gene to get the phoA-hEGF fused gene. It was synthesized and cloned into the pET23b vector with promoter T7 and terminator b0015, and transformed into E. coli Rosetta Strain.

Figure 1 Design of the phoA-hEGF.

Characterization

Figure2 Gel image of the phoA-hEGF.

Western blot proving the successful expression of hEGF

Figure3 The result of Western blto.

Western Blot results showed that there was a band of about 8 kDa in the extracellular and cellular contents of the bacteria cells, indicating the expression and secretion of phoA-hEGF fusion protein. The phoA signal peptide successfully guided the hEGF fusion protein into the periplasmic space.

Figure4 The successful expression of hEGF

From the figure, the level of concentration of pT7-hEGF group out of the cell is significantly lower than that of the intracellular group. However, the level of concentration of pT7-phoA-hEGF group is somewhat the same as the intracellular group, indicating that phoA signal peptide successfully guided the hEGF fusion protein into the periplasmic space.

Potential application directions

Our experiment proved that the phoA-hEGF fusion gene can effectively produce and secrete hEGF growth factor, and future research may focus on optimizing protein production and purification. At the same time, with this fusion gene, E. coli will secrete growth factors more efficiently, speeding up the repair of acne in patients, and reducing the cost of industrial production of this treatment.

Reference

1. Li X, et al. Construction and characterization of recombinant phoA-hEGF fusion protein: a promising therapeutic candidate. Protein Expr Purif. 2014; 94: 62-69. 2. Zhang Y, et al. Evaluation of the therapeutic effects of phoA-hEGF in a diabetic foot ulcer model. PLoS One. 2019; 14(6): e0219029. 3. Li G, et al. Identification of the key residues in phoA-hEGF responsible for its biological activity. Int J Biol Macromol. 2020; 164: 3272-3280. 4. Jiang H, et al. Increased secretion and purification of phoA-hEGF fusion protein in Escherichia coli using the YebF as a fusion partner. Protein Expr Purif. 2018; 144: 38-43. 5. Li X, et al. The potential applications of phoA-hEGF fusion protein in tissue engineering. Tissue Eng Part B Rev. 2016; 22(5): 459-470.

Sequence and Features