Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin

Abstract

The serine protease α-thrombin (FIIa) plays a fundamental role in blood clotting. In the present report, a FIIa precursor (pFIIa) was expressed in Nicotiana benthamiana Domin. The expression construct featured the Kozak consensus sequence and the 2S2 Arabidopsis thaliana (L.) Heynh. signal peptide to direct the protein into the secretory pathway (sec-pFIIa). A version carrying the KDEL endoplasmic reticulum (ER) retention signal (pFIIa-ER) was also constructed. Transient expression of pFIIa in N. benthamiana leaves was achieved by Agrobacterium tumefaciens infiltration. The influence of post-transcriptional gene silencing (PTGS) was analyzed by co-infiltrating with an A. tumefaciens strain carrying the construct for the Turnip Crinkle Virus-coat protein (TCV-CP) known for interfering with PTGS. Reverse transcription polymerase chain reaction and Western blot analyses confirmed the presence of the corresponding messenger RNA and the recombinant pFIIa protein in plant extracts. A positive effect of the addition of the PTGS inhibitor was demonstrated. The accumulation of sec-pFIIa and pFIIa-ER was estimated to be 6 μg g −1 fresh weight (FW) (0.07% (w/w) total protein concentration; TPC) and 17 μg g −1 FW (0.21% (w/w) TPC), respectively. Furthermore, stably transformed callus and suspension cultures were obtained. The recombinant protein was detected only in the biomass of the pFIIa-ER cell suspension line at a concentration of 0.25 μg mL −1 (0.017% (w/w) of total soluble protein). This appears to be the first report describing the expression of a precursor of FIIa in plants.