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Applied Biochemistry and Biotechnology

, Volume 174, Issue 4, pp 1558–1571 | Cite as

High Overexpression and Purification of Optimized Bacterio-Opsin from Halobacterium Salinarum R1 in E. coli

  • Fatemeh Abarghooi Kahaki
  • Valiollah BabaeipourEmail author
  • Hamid Rajabi Memari
  • Mohammad Reza Mofid
Article

Abstract

The purple membrane of Halobacterium Salinarum carries out a protein, bacteriorhodopsin (bR), which is a model for structure–function studies of membrane proteins. The heterologous expression of integral membrane proteins (IMPS) is difficult. In this study, we reported the heterologous overexpression of bacterio-opsin (bO) in Escherichia coli BL21 (DE3). Bacterio-opsin expression is facilitated by using mistic, a membrane protein from Bacillus subtilis in E. coli BL21 (DE3) membranes. The optimized bO gene was cloned in fusion to the C-terminus of mistic in pET 30a (+) and contains an oct-histidine in C-terminal to facilitate purification. Different medium, temperature, and induction time were used to optimize protein overexpression. The highest expression was obtained from the Terrific Broth (TB) medium at 18 °C with an IPTG concentration of 0.1 mM. The final purified bR was 192 ± 1 mg/L which has an important value for the production of membrane proteins in E. coli.

Keywords

Membrane protein Mistic Endogenous expression Bacterio-opsin 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Fatemeh Abarghooi Kahaki
    • 1
  • Valiollah Babaeipour
    • 1
    • 4
    Email author
  • Hamid Rajabi Memari
    • 2
  • Mohammad Reza Mofid
    • 3
  1. 1.Department of Life Science Engineering, Faculty of New TechnologiesUniversity of TehranTehranIran
  2. 2.Center of Biotechnology and Life Sciences and School of AgricultureShahid Chamran University of AhvazAhvazIran
  3. 3.Department of Biochemistry and Bioinformatics Research Center, School of Pharmacy and Pharmaceutical ScienceIsfahan University of Medical SciencesIsfahanIran
  4. 4.Department of Biological Science and BiotechnologyMalek Ashtar University of TechnologyTehranIran

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