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      Cloning, Expression, and Cost Effective Purification of Authentic Human Epidermal Growth Factor With High Activity

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          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.

          Objectives:

          The aim of this study is the cloning, expression, and purification of recombinant human EGF (rhEGF), and an assessment of its activity.

          Materials and 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.

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          Most cited references30

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          Molecular Cloning : A Laboratory Manual

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            Epidermal growth factor.

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              Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal.

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                Author and article information

                Journal
                Iran Red Crescent Med J
                Iran Red Crescent Med J
                10.5812/ircmj
                Kowsar
                Iranian Red Crescent Medical Journal
                Kowsar
                2074-1804
                2074-1812
                20 March 2016
                March 2016
                : 18
                : 3
                : e24966
                Affiliations
                [1 ]Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, IR Iran
                [2 ]Biology Research Center, Basic Sciences Faculty, Imam Hossein University (IHU), Tehran, IR Iran
                [3 ]Department of Chemistry, Basic Sciences Faculty, Imam Hossein University (IHU), Tehran, IR Iran
                Author notes
                [* ]Corresponding Author: Firouz Ebrahimi, Biology Research Center, Basic Sciences Faculty, Imam Hossein University (IHU), Tehran, IR Iran. Tel: +98-9123068466, Fax: +98-02177104934, E-mail: Febrhimi@ 123456ihu.ac.ir
                Article
                10.5812/ircmj.24966
                4884627
                27247796
                161b9658-3ed1-4b0c-80ef-532718fd6422
                Copyright © 2016, Iranian Red Crescent Medical Journal.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License ( http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

                History
                : 29 October 2014
                : 21 December 2014
                : 31 December 2014
                Categories
                Research Article

                Medicine
                epidermal growth factor,mtt,nih 3t3 cells
                Medicine
                epidermal growth factor, mtt, nih 3t3 cells

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