Volume 68, Issue 11 (4 2011)                   Tehran Univ Med J 2011, 68(11): 629-637 | Back to browse issues page

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M H, N D, F D, K M, F B, F M. Optimizing production of recombinant tissue plasminogen activator in non-pathogenic Leishmania by two genetic constructs. Tehran Univ Med J 2011; 68 (11) :629-637
URL: http://tumj.tums.ac.ir/article-1-289-en.html
1- , mahboudi.f@gmail.com
Abstract:   (5672 Views)

Background: Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agentsused in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae), a non-pathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins.
Methods: Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay.
Results: Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media,respectively.
Conclusion: The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems.

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