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Title

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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    APA: Copy

    POURANVARI, SARA, EBRAHIMI, FIROUZ, JAVADI, GHOLAMREZA, & MADDAH, BOZORGMEHR. (2016). CLONING, EXPRESSION, AND COST EFFECTIVE PURIFICATION OF AUTHENTIC HUMAN EPIDERMAL GROWTH FACTOR WITH HIGH ACTIVITY. IRANIAN RED CRESCENT MEDICAL JOURNAL (IRCMJ), 18(3), 0-0. SID. https://sid.ir/paper/293209/en

    Vancouver: Copy

    POURANVARI SARA, EBRAHIMI FIROUZ, JAVADI GHOLAMREZA, MADDAH BOZORGMEHR. CLONING, EXPRESSION, AND COST EFFECTIVE PURIFICATION OF AUTHENTIC HUMAN EPIDERMAL GROWTH FACTOR WITH HIGH ACTIVITY. IRANIAN RED CRESCENT MEDICAL JOURNAL (IRCMJ)[Internet]. 2016;18(3):0-0. Available from: https://sid.ir/paper/293209/en

    IEEE: Copy

    SARA POURANVARI, FIROUZ EBRAHIMI, GHOLAMREZA JAVADI, and BOZORGMEHR MADDAH, “CLONING, EXPRESSION, AND COST EFFECTIVE PURIFICATION OF AUTHENTIC HUMAN EPIDERMAL GROWTH FACTOR WITH HIGH ACTIVITY,” IRANIAN RED CRESCENT MEDICAL JOURNAL (IRCMJ), vol. 18, no. 3, pp. 0–0, 2016, [Online]. Available: https://sid.ir/paper/293209/en

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