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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 references 34

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      Epidermal growth factor.

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        Solubilization and refolding of bacterial inclusion body proteins.

        Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles. Refolding of inclusion body proteins into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost in production of recombinant proteins from E. coli. With new information available on the structure and function of protein aggregates in bacterial inclusion bodies, it has been possible to develop improved solubilization and refolding procedures for higher recovery of bioactive protein. Inclusion bodies are formed from partially folded protein intermediates and are composed of aggregates of mostly single types of polypeptide. This helps to isolate and purify the protein aggregates to homogeneity before solubilization and refolding. Proteins inside inclusion body aggregates have native-like secondary structures. It is assumed that restoration of this native-like secondary structure using mild solubilization conditions will help in improved recovery of bioactive protein in comparison to solubilization using a high concentration of chaotropic agent. Analysis of the dominant forces causing aggregation during inclusion body formation provides information to develop suitable mild solubilization procedures for inclusion body proteins. Refolding from such solubilized protein will be very high due to restoration of native-like secondary structure. Human growth hormone inclusion bodies were purified to homogeneity from E. coli cells before solubilization and refolding. Pure inclusion bodies were solubilized at alkaline pH in the presence of 2 M urea solution. The solubilized proteins were refolded using a pulsatile renaturation process and subsequently purified using chromatographic procedures. More than 40% of the inclusion body proteins could be refolded back to the bioactive native conformation. Mild solubilization is thus the key for high recovery of bioactive protein from inclusion bodies.
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          Epidermal growth factor.

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            Author and article information

            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
            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
            27247796
            4884627
            10.5812/ircmj.24966
            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.

            Categories
            Research Article

            Medicine

            nih 3t3 cells, mtt, epidermal growth factor

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