title: Effect of Culture Condition Variables on Human Endostatin Gene Expression inEscherichia coliUsing Response Surface Methodology.
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.
  
  
  OBJECTIVE
  This investigation aimed to predict and develop the optimal culture conditions for mRNA expression of the synthetic human endostatin (hES) gene inEscherichia coliBL21 (DE3).
  
  
  METHODS
  The hES gene was amplified, cloned, and expressed in theE. coliexpression 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 inE. coli.
authors: Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors: Abbas
authors: A
authors: Mohajeri
authors: Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran; Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors: Yones
authors: Y
authors: Pilehvar-Soltanahmadi
authors: Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors: Jalal
authors: J
authors: Abdolalizadeh
authors: Neurosciences Research Center (NSRC), Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors: Pouran
authors: P
authors: Karimi
authors: Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, IR Iran; Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, IR Iran.
authors: Nosratollah
authors: N
authors: Zarghami
keywords: Angiogenesis Inhibitors; Endostatin; Escherichia coli; Gene Expression