title: Cloning, Expression, and Cost Effective Purification of Authentic Human Epidermal Growth Factor With High Activity.
abstract: BACKGROUND
  Epidermal growth factor (EGF) plays a fundamental role in the healing of wounds relating to skin damage, the cornea, and the gastrointestinal tract.
  
  
  OBJECTIVE
  The aim of this study is the cloning, expression, and purification of recombinant human EGF (rhEGF), and an assessment of its activity.
  
  
  METHODS
  In the present experimental study, a synthetic pET28a (+) -hEGF construct was prepared. In order to ligate hEGF into pET24a (+), the PCR technique was performed, using special primers that possess restriction enzyme sites, which are also located in appropriate sites in pET24a (+). After transferring this construct into E. coli cells, protein expression was performed under standard conditions. Protein solubilization was done by urea. hEGF purification and refolding were carried out using gradient dialysis against the urea. We used RP-HPLC to compare between rhEGF and commercial rhEGF as a control. Finally, an MTT assay was performed to assess the viability of the NIH 3T3 cells treated with various concentrations of rhEGF.
  
  
  RESULTS
  Dialysis after urea solubilization caused precipitation of unwanted proteins, resulting in achievement of purified EGF with > 90% purity, without the need for expensive and time-consuming process. The MTT assay results showed that our rhEGF activate significantly higher proliferation of NIH 3T3 cells in comparison to the control (P-values were < 0.0001), in total concentrations and times evaluated
  
  
  CONCLUSIONS
  Via our purification protocol, a sufficient amount of bioactive recombinant human epidermal growth factor was obtained in just a few affordable steps, with superlative purity.
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Sara
authors.initials: S
authors.lastname: Pouranvari
authors.affiliation: Biology Research Center, Basic Sciences Faculty, Imam Hossein University (IHU), Tehran, IR Iran.
authors.forename: Firouz
authors.initials: F
authors.lastname: Ebrahimi
authors.affiliation: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Gholamreza
authors.initials: G
authors.lastname: Javadi
authors.affiliation: Department of Chemistry, Basic Sciences Faculty, Imam Hossein University (IHU), Tehran, IR Iran.
authors.forename: Bozorgmehr
authors.initials: B
authors.lastname: Maddah
keywords: Epidermal Growth Factor; MTT; NIH 3T3 Cells