Optimization of the Expression of Reteplase in Escherichia Coli Top10 Using Arabinose Promoter Publisher



Shafiee F¹ ; Moazen F¹ ; Rabbani M¹ ; Sadeghi HMM¹ Show Affiliations
Source: Jundishapur Journal of Natural Pharmaceutical Products Published:2015

Abstract

Background: Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. coli Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain of t-PA. Reteplase gene was ligated into pBAD/gIII plasmid which, allows secretion of this protein in periplasmic space. It would allow the correct formation of disulfide bonds in protein structure. Objectives: This study aimed at expression of reteplase in optimum condition. In this study, the reteplase gene was cloned and expressed in Escherichia coli top 10 as a suitable host cell and its expression was optimized. Materials and Methods: The recombinant plasmid, pET15b/reteplase was digested by NcoI and BamHI restriction enzymes; while pBAD/gIIIA vector was digested by NcoI and BglII. Then the insert and vector were ligated and used for transformation of E. coli Top10 cells by heat shock method. Overnight culture of transformed bacteria was induced by L-arabinose in various concentrations (0.2, 0.02, 0.002, and 0.0002%) and at various temperatures. Results: The obtained recombinant plasmid was sequenced to confirm the presence and correct framing of reteplase gene regarding the expression of reteplase. Maximum production of this enzyme was obtained under the following condition: 0.0002% L-arabinose at 37°C for 2 hours incubation. The purified protein was detected on SDS-PAGE (sodium dodecyl sulfate Polyacrylamide gel electrophoresis) as a 66 kDa band. The concentration of t-PA standard was 1 unit which is equal to 12 μg/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. Conclusions: Reteplase was expressed in E. coli Top 10 after activation of pBAD/gIIIA promoter region by arabinose and optimized. © 2015, School of Pharmacy, Ahvaz Jundishapur University of Medical Sciences.