• Indirect Optimization of Recombinant Protein Expression Levels in the Hsp27 Dicistronic SILEX System
  • Sana Razzazi,1 Fatemeh Sadat Shariati,2 Faezeh Takhsha,3 Arefe Sadat Khavari,4 Zahra Sedighi,5 Reza Ahangari Cohan,6,*
    1. Department of Biology , Faculty of Basic Siences , Shahed University , Tehran , Iran
    2. Infulenza Research Lab , Pasteur Institute of Iran , Tehran , Iran
    3. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
    4. Department of Biology , College of Basic Sciences , Shahed university , Tehran , Iran
    5. department of Medical Biotechnology , Faculty of Medicine, Shahed University, Tehran , Iran
    6. Department of Nanobiotechnology , NewTechnologies Research Group , Pasteur Institute of Iran ,Tehran ,Iran


  • Introduction: Design of experiments (DOE) is a statistical approach to plan, perform and interpret a large set of measurement data with a minimum number of experiments. The current study describes the optimization of a rapid screening system consisting of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), a protein model (staphylokinase, SAK) and a self-inducible system containing heat shock protein 27 (Hsp27).
  • Methods: Using RSM methodology, we indirectly identified the most effective factors on staphylokinase (SAK) expression levels among several variables, including inoculation rate, self-induction temperature and culture media, by fluorescence measurement of coupled eGFP (enhanced green fluorescent protein) expression in the dicistronic SILEX system. The expression level of SAK was measured in 25 different runs for 6 h incubation at 90 rpm.
  • Results: The results showed that all parameters had a significant effect (P < 0.05) on the expression level of SAK. The optimal expression conditions were an inoculation rate of 0.05, a temperature of 25°C and TB media. The analysis of variance coupled with the high value of R2 (0.91) showed that the quadratic model used for this prediction was highly significant (p < 0.05). Application of the optimized conditions resulted in a ~4-fold increase in SAK expression levels (from 1.3 to 5.2 µg/ml).
  • Conclusion: Furthermore, the recombinant SAK with a molecular mass of 16 kDa was purified by Ni-affinity chromatography and the activity was also confirmed by semi-quantitative caseinolytic assay.
  • Keywords: Expression, optimization, Escherichia coli, response surface methodology, green fluorescent protein