Nicotiana tabacum in vitro cultures: a promising plataform for producing recombinat human prethrombin

LAGUIA BECHER, M; ZALDÚA Z.; MARZOL, E.; MARCONI, P.; VELANDER, W.; ALVEREZ, MA.

Providence

Congreso; 2013 in vitro biology meeting; 2013

Society for in vitro biology

Active human prothrombin, a glycoprotein involved in blood coagulation, has been expressed in CHO and BHK cells. Plants (including in vitro cultures) are gaining attention for the production of recombinant proteins. Its advantages include that they can rapidly be bulked to large biomass, its relatively inexpensive maintenance, and that they do not harbor mammalian proteins or pathogens. We have expressed prethrombin-2 (FII), the smallest single-chain precursor to thrombin, in Nicotiana tabacum. We have designed two constructs bearing the human FII coding sequence (864 bp), under the control of the CaMV35S promoter, fused to the signal peptide 2S2 from A. thaliana to deliver the protein to the secretory pathway, the Kozak sequence, and with or without adding the C-terminal KDEL sequence for retrieving the protein to the endoplasmic reticulum (pFII/-A/RE). Those constructs were introduced into the plant expression binary vectors pK7WG2 and p35SGAT (pK7WG2:pFII-A/RE and p35GAT:pFII-A/RE). Then, they were introduced by electroporation into Agrobaterium tumefaciens that was used to agroinfiltrate N. tabacum young plants. To analyze if post-transcriptional gene silencing (PTGS) could interfere with FII expression a co-agroinfiltration in the presence of Turnip crinkle virus capside protein (CP-TCV) was performed. The Western blot analysis performed in plant extracts confirmed the protein integrity, showing a specific band of approximately 35 kDa in the two ER versions but, surprisingly only in the presence of the PTGS CP-TCV suppresor in the apoplastic versions. Our preliminary assessment is that the recombinant pre- FII here expressed is in the proper molecular weight range and structure needed for coagulation function.