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Covalent-display of an active chimeric-recombinant tissue plasminogen activator on polyhydroxybutyrate granules
Akram Hafizi1,2, Mohamad Ali Malboobi3, Mokhtar Jalali-Javaran1
1Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran.
Objective:
To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes.
Results:
Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA.
Conclusion:
Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.
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