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Repository of Research and Investigative Information

Ahvaz Jundishapur University of Medical Sciences

Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology

Mohajeri, Abbas and Pilehvar-Soltanahmadi, Yones and Abdolalizadeh, Jalal and Karimi, Pouran and Zarghami, Nosratollah (2016) Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology. Jundishapur Journal of Microbiology, 9 (8). ISSN 2008-3645

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Official URL: http://doi.org/10.5812/jjm.34091

Abstract

Background: Recombinant human endostatin (rhES) is an angiogenesis inhibitor used as a specific drug for the treatment of non-small-cell lung cancer. As mRNA concentration affects the recombinant protein expression level, any factor affecting mRNA concentration can alter the protein expression level. Response surface methodology (RSM) based on the Box-Behnken design (BBD) is a statistical tool for experimental design and for optimizing biotechnological processes. Objectives: This investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene in Escherichia coli BL21 (DE3). Materials and Methods: The hES gene was amplified, cloned, and expressed in the E. coli expression system. Three factors, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, post-induction time, and cell density before induction, were selected as important factors. The mRNA expression level was determined using real-time PCR. The expression levels of hES mRNA under the different growth conditions were analyzed. SDS-PAGE and western blot analyses were carried out for further confirmation of interest-gene expression. Results: A maximum rhES mRNA level of 376.16% was obtained under the following conditions: 0.6 mM IPTG, 7 hours post-induction time, and 0.9 cell density before induction. The level of rhES mRNA was significantly correlated with post-induction time, IPTG concentration, and cell density before induction (P < 0.05). The expression of the hES gene was confirmed by western blot. Conclusions: The obtained results indicate that RSM is an effective method for the optimization of culture conditions for hES gene expression in E. coli.

Item Type: Article
Keywords: Endostatin; Angiogenesis Inhibitors; Gene Expression; Escherichia coli
Subjects: R Medicine > R Medicine (General)
Depositing User: آقای بهرام سوسنی غریب وند
Date Deposited: 13 Nov 2016 06:21
Last Modified: 13 Nov 2016 06:21
URI: http://eprints.ajums.ac.ir/id/eprint/2609

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