Rapid Screening for High Expressing Multicopy Recombinants and Enhanced Epidermal Growth Factor (EGF) Protein Production Using Pichia Pastoris

Epidermal growth factor (EGF) is a highly effective stimulator which promotes cell proliferation and this has led it to being proposed as a prospective healing agent for various treatments of wounds and many other clinical applications. With such a high demand for this protein, it is therefore important to firstly develop an expression system that efficiently generates sufficient amounts of the protein but also of high purity and biological activity. In this study, we have developed an efficient recombinant EGF production route that includes rapid multicopy recombinant screening using Pichia pastoris. In addition, we have optimized protein expression conditions and a simple protein purification procedure. Firstly, quantification of the ratio of hEGF PCR band brightness to AOX1 was used to screen the multicopy transformants. Furthermore, Real-time PCR was used to detect the copy number of hEGF integrated into the Pichia pastoris genome, which is consistent with the normal PCR results. Optimization of recombinant hEGF expression using the selected multicopy Pichia pastoris strain was performed, hEGF protein was highly expressed under pH 7 conditions for a culture period of 60 h. Moreover, ammonium sulfate precipitation in combination with cationic affinity and hydrophobic interaction chromatography were used to produce 99% pure recombinant hEGF, with high biological activity of 2 × 10 6 IU/mg. Thus we have developed a method using Pichia pastoris to produce high levels of hEGF of high purity and specific activity. Our route may also be suitable for the production of other therapeutic recombinant proteins.