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.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.

Isoprocurcumenol Supports Keratinocyte Growth and Survival through Epidermal Growth Factor Receptor Activation

“…Although EGF seems to be an attractive molecule for both therapeutic and skin cosmetic applications, there are several hurdles to overcome before its usage can be commercialized. Firstly, due to the problem with low yield of EGF from human sources, protein synthesis and purification of EGF is very expensive [15]. Additionally, since EGF is a protein, it is very difficult to maintain and control its activity [16].…”

Development of a Fast Organic Extraction–Precipitation Method for Improved Purification of Elastin-Like Polypeptides That Is Independent of Sequence and Molecular Weight

“…A lower molecular weight ELP was selected for testing our extraction–precipitation method since sequences of this length have a limited ability to transition effectively via ITC. Furthermore, optimized purifications of EGF from E. coli frequently include urea to assist in protein solubilization; , however, the use of organic solvent extraction obviates this step to enable direct extraction from E. coli . A binding experiment was performed using MB49 murine bladder cancer cells to test for EGF binding activity after purification by extraction–precipitation.…”

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“…Taking advantage of the lenient approval and monitoring activities in cosmetic and skin care industries, rEGF isoforms have been applied commercially to result in a wide collection of marketable end products, irrespective of the absence of documentation of their origins and structural properties [40][41]. Obviously and understandably, the feedbacks received from the customers or end users, have been highly controversial.…”