Cloning of Tissue Plasminogen Activator cDNA inNonpathogenic Leishmania

Cloning of Tissue Plasminogen Activator cDNA in Nonpathogenic Leishmania

Introduction: At present, most recombinant proteins are produced inprokaryotes especially E coli. Yeasts and CHO also are used aseukaryotic hosts. Leishmania tarentolae, a parasite of lizards, amember of Trypanosomatidae family is one of the new systems forexpression of heterologous proteins. In this system, some of theparasitic protozoa features are used in expression of mammalianproteins.Material and Methods: For evaluation of the protozoa forexpression of human complex proteins, we cloned cDNA of tPA genecontaining native human signal sequence. We used vectorscontaining 3´ and 5´ sequences of Leishmania 18s rRNA forintegration of the vectors in 18s rRNA gene and severe transcription.Results: RT-PCR test showed production of specific mRNA of tPAgene in the recombinant cells. Southern blot analysis confirmed thecloning of t-PA in the genome of the Leishmania.Conclusion: This study showed native human signal sequencemediate transport and secretion of the protein. Hence, L. tarentolaeis the first useful biotechnologically protozoan and tPA is the mostcomplex protein expressed in it.

Introduction: At present, most recombinant proteins are produced in prokaryotes especially E coli. Yeasts and CHO also are used as eukaryotic hosts. Leishmania tarentolae, a parasite of lizards, a member of Trypanosomatidae family is one of the new systems for expression of heterologous proteins. In this system, some of the parasitic protozoa features are used in expression of mammalian proteins. Material and Methods: For evaluation of the protozoa for expression of human complex proteins, we cloned cDNA of tPA gene containing native human signal sequence. We used vectors containing 3ʹ and 5ʹ sequences of Leishmania 18s rRNA for integration of the vectors in 18s rRNA gene and severe transcription. Results: RT-PCR test showed production of specific mRNA of tPA gene in the recombinant cells. Southern blot analysis confirmed the cloning of t-PA in the genome of the Leishmania. Conclusion: This study showed native human signal sequence mediate transport and secretion of the protein. Hence, L. tarentolae is the first useful biotechnologically protozoan and tPA is the most complex protein expressed in it.