Modification of Recombinant Human Epidermal Growth Factor (rh-EGF) Expression Vector by Site Directed Mutagenesis for Therapeutic Protein Production

Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency related 10 diseases. The recombinant protein was designed to be expressed in pET21b(+) vector using Escherichia coli 11 BL21(DE3) expression host. However, in our previous study, the gene of rh-EGF was constructed aside gene of 12 6xHisTag without any restriction sites, so it is not possible to obtained a purified and single rh-EGF. In this study, 13 we modified the rh-EGF expression vector with a common method, namely site-directed mutagenesis (SDM) to 14 remove the gene of 6xHisTag. The vector modification was carried out by inserting stop codons and EcoR1 15 restriction site, also deleting 6xHisTag sequence with PCR-based SDM. The results of PCR showed non-specific 16 bands, PCR 2-step cycles produced one non-specific band and PCR 3-step cycles produced two non-specific 17 bands. All of the PCR products were purified by gene isolation. The SDM-recombinant plasmids which were 18 treated for template plasmid-free product were transformed to E.coli DH5α. The result of transformation had low 19 transformant efficiency score. However, gene mutation with deletion of 6xHisTag and insertion of stop codons 20 and EcoRI restriction site in plasmid pET21b(+) had been successfully done. In addition, in the expression level, 21 it is proven that the modified vector results rh-EGF which has similar size with rh-EGF standard and 22 approximately 1 kDa smaller than that of our previous rh-EGF-6xHisTag. 23