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. 2016 May 8;9(8):e34091.
doi: 10.5812/jjm.34091. eCollection 2016 Aug.

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

Abbas Mohajeri  1 , Yones Pilehvar-Soltanahmadi  2 , Jalal Abdolalizadeh  3 , Pouran Karimi  4 , Nosratollah Zarghami  2
Affiliations

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

Abbas Mohajeri et al. Jundishapur J Microbiol. .

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.

Keywords: Angiogenesis Inhibitors; Endostatin; Escherichia coli; Gene Expression.

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Figures

Figure 1.
Figure 1.. Amplification of hES Coding Sequence and Construction of Expression Vector
A, Amplification of hES coding sequence using Pfu DNA polymerase. Lane 1: gene ruler DNA ladder mix (thermo scientific); lane 2: negative control; lane 3: amplified ORF of hES (592 bp). B, Construction of the pET26b-hES vector.
Figure 2.
Figure 2.. Diagnostic Plots for Estimating the Adequacy of the Regression Model
A, Correlation between predicted and actual values for hES mRNA expression; B; the studentized and normal percentage probability plot of hES mRNA expression.
Figure 3.
Figure 3.. Response Surface Plots (left) and Contour Plots (right) of rhES mRNA Revealed the Relationship Between the Response and Actual Levels of Each Independent Variable
A, The effect of inducer concentration (IPTG) and post-induction time on rhES mRNA; B, the effect of inducer concentration (IPTG) and cell density before induction time on rhES mRNA; C, the effect of post-induction time and cell density before induction time on rhES mRNA.
Figure 4.
Figure 4.. SDS-PAGE and Western Blot Analysis of Expressed hES Gene
A, SDS-PAGE (12%); B, Western blot. Lane 1, protein marker; lane 2, total protein before induction; lane 3, total protein after induction in recombinant pET26b.
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