Home Bratislava Medical Journal 2019 Bratislava Medical Journal Vol.120, No.10, p.757-763, 2019

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Published Monthly, in English
Founded: 1919
ISSN 0006-9248
(E)ISSN 1336-0345
 

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Bratislava Medical Journal Vol.120, No.10, p.757-763, 2019

Title: High expression level of human epidermal growth factor (hEGF) using a well-designed fusion protein-tagged construct in E. coli
Author: D. Shams, M. Alizadeh, S. Azari, S. Hosseini, S. Yasami-Khiabani, S. Samani, M. A. Shokrgozar

Abstract: AIM: The study was aimed at design a good fusion construct that would successfully express the recombinant proteins and produce peptides in Escherichia coli. Two different constructs including human epidermal growth factor (hEGF) gene were designed to obtain an efficient expression level of hEGF. The hEGF sequence was inserted in pET32a vector containing thioredoxin (Trx) sequence and modified pET15b vector containing intein and elastin-like polypeptide (ELP).
METHODS: The vectors were transformed into E. coli TOP10F′ for multiplication and further into E. coli BL21 (DE3) to express protein. The hEGF expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) while the expression levels were evaluated by SDS-PAGE and western blotting and compared by ImageJ analysis, BCA and Elisa assays.
RESULTS: The expression level after 2 hours of IPTG induction was significantly higher than after other induction times. ImageJ, BCA and Elisa analyses demonstrated that the Trx presence enhanced protein expression significantly when compared to ELP-intein-based construct.
CONCLUSION: The pET32a-Trx-hEGF construct had a higher expression than pET15b-ELP-intein-hEGF. Overall, considering Trx, the fusion protein in construct design can make it suitable to significantly express hEGF compared to ELP-intein while its combination with ELP-intein may improve the expression of the ELP-intein construct (Tab. 2, Fig. 7, Ref. 34).

Keywords: human epidermal growth factor (hEGF), thioredoxin (Trx), elastin-like polypeptide (ELP), intein, expression
Published online: 28-Oct-2019
Year: 2019, Volume: 120, Issue: 10 Page From: 757, Page To: 763
doi:10.4149/BLL_2019_126


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