Production and purification of constructed recombinant hirudin in BL21(DE3) strain

Abstract

Background/Objective: Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway, it is the most potent natural inhibitor of thrombin, it binds thrombin with high affinity, so, the aim of this study was to build hirudin gene by overlapping extension PCR then cloning and expression in BL21(DE3) strain. Methods: Hirudin gene constructed with four modified primers then the final product amplified by two primers named as A, B by using overlapping extension PCR, for gene expression, BL21(DE3) strain was used under the control of T7 promoter in pET-16b vector and for hirudin production, LB broth medium was used as fermentation medium, to detect the expression of hirudin in BL21(DE3) strain in fermentation media real-time PCR was used. Hirudin protein purified at first by (Immobilized metal affinity chromatography) IMAC, then this protein was dialysis and treated with factor Xa to eliminate His-tag. Then hirudin purified by DEAE sepharose and SP sepharose column, the concentration of protein determined by ELISA, furthermore the activity evaluated by thrombin titration and activated partial thromboplastin time (APPT) test. Results: Hirudin gene constructed in two round PCR first round produced two products (product 1,117 bp while product 2,114 bp) the second-round PCR gave the final product 213 bp. The resulted band from gel electrophoresis for constructed vector pET-16b-HirudinS was (5,901 bp). Hirudin expression was established by real-time PCR with Ct value (20.28). The analysis on 15% SDS-PAGE for the SP sepharose column illustrated the hirudinS protein band with size about ∼10.8. Concentration of produced hirudin within its solution reached to 1.75 ng, thrombin titration method showed that the hirudin protein required 300 µl from thrombin to clot, also, APPT test showed that hirudin elongated clotting time to 7 min in comparison with 6min for aspirin and the statistical analysis results for APPT test illustrated that there was no significant difference between hirudin and aspirin. Conclusion: This study approved that overlapping extension PCR is a good strategy for building hirudin gene and it's successfully expressed in BL21(DE3) strain.

Cell-free synthesis of the hirudin variant 1 of the blood-sucking leech Hirudo medicinalis

“…In addition, a cell-free synthesis approach with rabbit reticulocyte lysate was described as well 20 . Commercially available hirudin variants like Desirudin and the discontinued Lepirudin are nowadays expressed in Saccharomyces cerevisiae 21 – 23 , whereas for research applications usually protocols based on E. coli or Pichia pastoris are applied 7 , 23 – 25 .…”