Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae.
A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, 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 the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA 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 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 the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.
Soleimani
Mohammad
M
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Mahboudi
Fereidoun
F
Davoudi
Noushin
N
Amanzadeh
Amir
A
Azizi
Mohammad
M
Adeli
Ahmad
A
Rastegar
Hossein
H
Barkhordari
Farzaneh
F
Mohajer-Maghari
Behrokh
B
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D007891:Leishmania / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator