Optimization of the Expression of Reteplase in Escherichia coli TOMO Using Arabinose Promoter

Abstract

Background: Reteplase is a mutant version of t-PA (tissue plasminogen activator) with prolonged half-life. In the present study, E. colt Top 10 bacteria were utilized in the production of reteplase, which is the nonglycosylated active domain oft-PA. Reteplase gene was ligated into pBAD/gIll 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 colt top 10 as a suitable host cell and its expression was optimized. Materials and Methods: The recombinant plasmid, pET15b/reteplase was digested by Ncol and BumHI restriction enzymes; while pBAD/ glIIA vector was digested by Ncol and BglII. Then the insert and vector were ligated and used for transformation of E. co/iTopio 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 plasm id 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 degrees 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 mu g/mL. The enzymatic activity of samples was measured as 0.8 units compared to the standards. Conclusions: Reteplase was expressed in E. coliTop 10 afteractivation of pBAD/gIllA promoter region by arabinose and optimized.