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Abstract

Purification and biological activity assessment: Comparison between two recombinant human epidermal growth factors with different molecular weights

Considering its physiological roles in repair of skin damages, cornea and gastrointestinal tracks, epidermal growth factor has attracted scientist’s attention among other members of the growth factor family. Many investigations were done for recombinant production of this protein and all of them were focused on the methods of its purification and biological activity assessment. In this study, human epidermal coding gene was prepared as a synthetic construct between T7-tag and His6-tag in pET21a(+). After designing special primers for this gene, it was amplified by PCR technique and in order to making sticky ends, enzymatic digestions were done and then PCR product was ligated in pET28a(+). After transforming these construct into host cells, protein expression for both of them were done under standard condition. Protein solublization was performed by guanidine hydrochloride. hEGF purification from pET21(+)-EGF was done by affinity chromatography and to wash recombinant hEGF from pET28(+)-EGF, sodium deoxycholat salt and passing them from Amicon filters was used. Significant comparison between produced cases and standard sample were performed by RP-HPLC and ultimately, biological activity assessment for them was performed by MTT standard test. Results either in purification method or in biological activity assessment were benefited to recombinant hEGF protein produced in the present study


Author(s): Mostafa Bakhshi, Firouz Ebrahimi*, Abbas Hajizadeh and Hani Keshavarz Alikhani

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