Purification and cell-based functional assay of recombinant human epidermal growth factor

Sara Pouranvari,1,* Mahdieh sadat taghavi,2 Firuz ebrahimi,3

1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
3. Biology Research Center, Basic Science Faculty, IHU, Tehran, Iran

Abstract

Introduction

Human epidermal growth factor (hegf) is a thermo stable 53 amino acid residue polypeptide with various medical and cosmetic applications such as wound healing. scientists have used recombinant dna technology to produce hegf efficiently. following this development, a variety of hosts (prokaryotic and eukaryotic), vectors, and purification methods were used. then, investigators checked the activity of recombinant hegf (rhegf) via different tests. if e.coli cells is used as host, recombinant human epidermal growth factor is expressed as inclusion body.

Methods

In present study, after transferring recombinant vector pet24a (+) into e. coli cells, protein expression was done under standard conditions. recombinant human epidermal growth factor was expressed as inclusion body. purification of inclusion bodies was performed using detergent washing with sodium deoxicholate. we try to solubilize inclusion bodies using urea (8 molar) and refolding were carried out using gradient dialysis against the urea (4 m, 2 m, 1 m, and 0 m). after dialysis a single band of rhegf was observed in sds page. the final purified rhegf was quantified by rp-hplc. finally, to ensure that rhegf had been properly folded and was therefore functionally active, mtt assay was performed by nih 3t3 cell line. growth-promoting activity was monitored on both day 1 and day 3.

Results

Solubilization and purification of rhegf were performed properly. rp-hplc chromatogram showed a main single peak. this peak was related to rhegf, as it completely matched commercial rhegf. mtt assay results showed cell viability of our rhegf were significantly (p-values were < 0.0001) higher than the control.

Conclusion

In conclusion, via our purification protocol, a sufficient amount of bioactive rhegf was obtained in few steps with superlative purity. it seems, proper folding of rhegf in purification process leads to higher proliferation of nih-3t3 cells compared with control.

Keywords

Recombinant human epidermal growth factor, refolding, mtt assay, nih 3t3 cell line