Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 
  • Users Online: 431
  • Home
  • Print this page
  • Email this page
ORIGINAL ARTICLE
Year : 2019  |  Volume : 14  |  Issue : 6  |  Page : 554-565

Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein


1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran
2 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences; Protein Technology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran

Correspondence Address:
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-5362.272564

Rights and Permissions

Recombinant epithelial cell adhesion molecule extracellular domain (EpEX) has a high potential as a candidate for passive and active immunotherapy as well as cancer vaccination. In the present study, EpEX was expressed as a thioredoxin fusion protein in Escherichia coli (E. coli). The effect of different hosts and expression conditions on the expression level of the fusion protein was also evaluated. Moreover, the effect of temperature and isopropyl-ß-d-thiogalactopyranoside (IPTG) concentration on protein solubility was assessed. The codon optimized-synthetic gene was cloned into pET32a (+) expression vector and transformed into E. coli BL21 (DE3), RosettaTM (DE3), and OrigamiTM (DE3). The protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Lowering the expression temperature to 16 °C and IPTG concentration to 0.5 mM also dramatically increased the volumetric productivity of the fusion protein. In optimum culture condition, high-level expression of the target fusion protein was detected in RosettaTM (DE3) and OrigamiTM (DE3) (207 and 334 μg/mL, respectively), though they were expressed as inclusion bodies. No improvement was observed in the solubility of the fusion protein by reducing the temperature or IPTG concentration even when expressed in a TrxB/gor mutant strain. Results showed that Trx tag combined with other strategies utilized here could be effective to achieve high level of protein production but not effective in solubility improvement. However, new approaches might be necessary to enhance the solubility of EpEX in the E. coli system.


[FULL TEXT] [PDF]*
Print this article     Email this article
 Next article
 Previous article
 Table of Contents

 Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
 Citation Manager
 Access Statistics
 Reader Comments
 Email Alert *
 Add to My List *
 * Requires registration (Free)
 

 Article Access Statistics
    Viewed267    
    Printed13    
    Emailed0    
    PDF Downloaded63    
    Comments [Add]    
    Cited by others 1    

Recommend this journal