Expression of the recombinant plasminogen activator (reteplase) by a non-lytic insect cell expression system

S Aflakiyan,H Mir Mohammad Sadeghi, M Shokrgozar, M Rabbani, S Bouzari, A Jahnian-Najafabadi

Abstract


Reteplase is a potent thrombolytic agent which is widely used in the management of acute myocardial infarction and stroke. It belongs to the third generation of the thrombolytic drugs and has been derived from native human tissue plasminogen activator by removing three domains of it and keeping the Kringle 2 and Serine protease domains. However, the high cost of this drug, has limited the application of this drug especially in the developing and third world countries. The most laborious steps in the bacterial production of this drug is its purification and refolding steps which keep the process yield low and the cost high. Therefore, in the present study we evaluated the expression of reteplase by a non-lytic insect cell expression system. Following cloning and transfection procedures, recombinant Sf9 insect cell clones expressing the reteplase protein were selected. Primarily, the expression was verified by dot-blot analysis and subsequently it was confirmed by Western Blotting showing a band of about 45 kD on nitrocellulose membrane. The biological activity of the expressed protein was also evaluated and showed to be about 29 IU/ml. This confirmed the possibility of expression and the correct folding of the expressed protein. Hence, optimization of the expression followed by purification of the protein could be the next steps of the study.


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