Transient expression of an active Toxoplama gondii serine proteinase inhibitor in tobacco leaves

LAGUÍA BECHER M; DUSCHAK V G; CLEMENTE M

Rosario-Argentina

Simposio; REDBIO; 2009

REDBIO-Argentina

cDNA of Toxoplasma gondii kazal proteinase inhibitor (TgPI-124-294) was cloned for the first time into a plant expression vector with an apoplastic signal peptide and was transiently expressed in Nicotiana tabacum leaves through Agrobacterium mediated gene transfer by vacuum infiltration method. Expression of TgPI-124-294 was confirmed by Western blot using a polyclonal anti-TgPI-124-294 antibody and the expression level was determined to be 0.5 % of total soluble proteins of apoplastic washing fluid (AWF) of tobacco leaves. In vitro bioactivity determination of plant expressed TgPI-124-294 was performed by analysis of the relative inhibitory activity of increasing amounts of TgPI-124-294-derived plant extracts versus different serine proteinases. AWF-TgPI-124-294 presented inhibitory activities towards animal trypsin (75 %), chymotrypsin (50 %), thrombin (85 %) and human neutrophil elastase (40 %). In addition, the TgPI-1 potential biotechnology applications are discussed.