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Tissue plasminogen activator (tPA) is a 65 kDa member of serine protease family. tPA is naturally expressed by endothelial cells of vessels in negligible amounts. The enzyme converts plasminogen to plasmin and results in dissolution of blood clots. Retepalse (Retavase) is a mutated variant of human tPA that lacks a segment of 137 residues. Reteplase is produced in Escherichia coli and lacks post translational modifications including glycosylation. Due to presence of nine disulfide bonds, expression of this protein within bacterial systems is so difficult and mostly results in protein aggregates. Refolding and reactivation of inclusion bodies is an expensive, time-consuming procedure with low efficacy for disulfide-bonded proteins. In the present research we have changed the regulatory sequences to produce active and soluble reteplase enzyme in E. coli BL21 host. Inducible expression of reteplase under the control of T7 promoter resulted in aggregation of proteins in inclusion bodies while the use of a constitutive promoter could produce biologically active reteplase in the cytosol of E. coli cells.

Keywords: reteplase, tissue plasminogen activator, constitutive expression, inducible expression, active form

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