Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.16

PRODUCTION OF RECOMBINANT HUMAN THROMBOPOIETIN IN TRANSGENIC TOBACCO PLANTS

DE AMICIS F.*, PATTI T.**, DAMIANI D.***, FOGHER C.****, MARCHETTI S.*

*) DPVTA, Università di Udine, Via delle Scienze 208, 33100 Udine

**) TransPharma srl, AREA Science Park, Padriciano 99, 34012 Trieste

***) DRMM, Università di Udine, Via delle Scienze 208, 33100 Udine

****) Istituto di Botanica e Genetica Vegetale, Università Cattolica, Via Emilia Parmense 84, 29100 Piacenza

thrombopoietin, megakaryocytopoiesis, tobacco, pharma production

Thrombopoietin (TPO) is a hematopoietic cytokine that primarily regulates megakaryocytopoiesis and platelet production; it was shown to decrease the need for platelet transfusion and attenuate chemotherapy-induced thrombocytopenia. At present, TPO is produced by mammalian cell culture in limited amounts and at a very high cost. For pharma production, transgenic plants offer an attractive alternative to both microorganisms and animals.

In this work, we have isolated the complete coding sequence of human TPO from a liver cDNA library. After cloning and sequencing, the TPO cDNA has been inserted in a plant expression vector under the control of the enhanced 35S CaMV promoter and downstream an artificial 5’ UTR causing a 20-fold increase of transgene expression levels in tobacco mesophyll. In order to shuttle the recombinant protein into the lumen of endoplasmic reticulum for glycosylation and subsequent secretion to the apoplast, the transit peptide sequence of human TPO was mantained.

Several transgenic tobacco (N. tabacum, cv. Xanthi) plants have been produced through Agrobacterium-mediated transformation. These plants are currently under evaluation with M-07e, a human TPO-dependent megakaryoblastic leukemia cell line expressing the human TPO receptor. With the ultimate objective to ease purification of recombinant TPO from plant tissues, a synthetic peptide corresponding to an essential domain of the human cytokine has been used to raise antibodies in rabbit. Once the production levels and the activity of the plant-derived TPO will be assessed, transgenic plants will be crossed with tobacco lines harbouring genes enabling glycan conversion to the human type.