Expression of recombinant human prethrombin-2 in Nicotiana tabacum

MELINA LAGUÍA BECHER; ELIANA MARZOL; PATRICIA MARCONI; MARÍA ALEJANDRA ALVAREZ

Mar del Plata

Congreso; VIII Encuentro Latinoamericano y del Caribe de Biotecnología - REDBIO ARGENTINA 2013 y IX Simposio Nacional REDBIO; 2013

REDBIO

The expression of human prethrombin-2 (FII), the smallest single-chain precursor of thrombin, was evaluated in N. tabacum. FII gene was cloned under the control of the Cauliflower Mosaic Virus 35S promoter (CaMV 35S) with or without adding the 5? leader sequence (§Ù) of Tobacco Mosaic Virus (TMV). The expression of the recombinant FII was directed to either the apoplast (FII-A) or the endoplasmic reticulum (FII-RE). All the FII sequences were transiently expressed in tobacco leaves by agroinfiltration and analyzed by Western blot in the protein extracts. A specific band of 35 kDa, corresponding to the predicted molecular weight of FII, was detected in all extracts from ER-tagged constructions but it was absent in FII-A extracts. The co-expression with the capsid protein from Turnip Crinkle Virus (TCV-CP), which is known to act as a suppressor of gene-silencing, increased the expression of all constructions allowing the detection of apoplastic-targeted proteins. The highest levels of FII expression were obtained with the §Ù enhancer in combination with the TCV-CP that produced 0.25 % and 0.08 % of total protein content for the 35S§ÙFII-ER and the 35S§ÙFII-A genes, respectively. These results demonstrate that plants can be used as alternative expression systems to produce thrombin.