Nicotiana tabacum as an alternative production platform for recombinant prethrombin

LAGUÍA BECHER, M; MARCONI, P; RICCO, VALERIA; ZALDÚA, Z; VELANDER, W; ALVAREZ, MA

Viena

Simposio; International Symposium Natural Products And Drug Discovery - Future Perspectives; 2014

University of Innsbruck

Plants (both whole plants and in vitro cultures) are gaining attention for large-scale production of recombinant proteins for health although more research has to be performed in order to gain general acceptance. Some of the advantages of plants as recombinant protein production platforms include that they can rapidly be bulked to large biomass, its relatively inexpensive maintenance, and that they do not harbor mammalian proteins or pathogens. To date, there is more than a dozen of plant-made biopharmaceuticals in clinical developments [1]. Protein production could be achieved through stable or transient expression. The stable transformation methods, which are generally in use, modify the genetic background of the productive plant species generating biosafety concerns [2,3]. The transient expression strategy, where the foreign DNA is not integrated into the plant genome, has a much shorter timeframe than nuclear or plastid transformation with yields that are in general higher to those obtained when a stable transformation is performed. Also, it has the ability to meet the stringent demands for high quality biologics and high biosafety standards at a competitive scale and cost [4,5]. Active human prothrombin, a glycoprotein involved in blood coagulation, has been expressed in CHO and BHK cells.Thrombine is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex [6]. We have expressed prethrombin-2 (FII), the smallest single-chain precursor of 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, and with the Kozak sequence. One of the constructs also have the C-terminal KDEL sequence for retrieving the protein to the endoplasmic reticulum (pFII/-A/RE). The constructs were cloned into the plant expression binary vectors pK7WG2 and p35SGAT (pK7WG2:pFII-A/RE and p35GAT:pFII-A/RE) which were introduced by electroporation into Agrobaterium tumefaciens. Leves of N. tabacum young plants were agroinfiltrated with the transformed A. tumefaciens strains, and leave samples were taken every day during a week to analyze if any recombinant FII was tansiently expressed. 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 also performed. FII was expressed in N. tabacum leaves. The Western blot analysis 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 conformational structure needed for the coagulation process.