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Biotechnol. Appl. Biochem. (2007) Immediate Publication, doi:10.1042/BA20060217 
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| Expression of human tissue-type plasminogen activator (t-PA) in Leishmania tarentolae |
| Mohammad Soleimani, Fereidoun Mahboudi, Noushin Davoudi, Amir Amanzadeh, Mohammad Azizi, Ahmad Adeli, Hossein Rastegar, Farzaneh Barkhordari and Behrokh Mohajer-Maghari |
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| Biotechnology Research Center, Pasteur Institute of Iran, TEHRAN, Iran.
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A variety of Recombinant protein expression systems have been developed for heterologous gene in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammalian, insects, transgenic animals and transgenic plant. Also, it has been reported that Leishmania tarentolae (Tarentolae anularis) a parasite of lizard has the capability for expression of heterologous genes. Trypanosomatidae are rich in glycoproteins which can account for more than 10% of total protein. The oligosaccharide structures of their glycopropteins are similar to those of mammalian with N-linked galacotse, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In this study we tried to express tissue plamsinogen activator (t-PA) gene in L. tarentolae. This protein is a thrombolytic agent with 527-amino-acid residues. t-PA possesses serine protease activity with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette including the alpha intergenic regions of Leishmania major (L. major) and two sites at the 3? and 5? ends for homologous recombination in L. tarentolae, in addition to antibiotic resistant genes. Southern blot analysis showed that human t-PA gene has been inserted into the genome of the parasite. The expression of the t-PA at the mRNA and protein level was confirmed. It was shown that the expressed t-PA in this system is 70 IU/ ml of culture media which is much higher than those reported previously in other systems.
doi:10.1042/BA20060217
Received 2 November 2006/20 February 2007; Accepted 20 March 2007
Published as Immediate Publication 20 March 2007
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