The expression, localization and characterization of recombinant trichosanthin, a type I ribosome-inactivating protein, in transgenic tobacco

Ribosome-inactivating proteins (RIPs) are adenine-specific rRNA N-glycosidases that inactivate eukaryotic and prokaryotic ribosomes. In addition, the RIPs have a variety of other enzymatic and antiviral functions, which vary by protein type and plant host. RIPS, and particularly the type I RIP trichosanthin (TCS) have recently aroused a great deal of interest for their abilities to confer viral resistance in plant and animal systems, particularly in the construction of transgenic plants and immunotoxins, and as a functional therapeutic for HIV and cancer. There is a strong focus, therefore, on the development of suitable expression systems for type I RIPS in plant systems.;In developing an expression system for RIPs in transgenic plants, we chose the type I RIP trichosanthin as the model protein. Trichosanthin (TCS) is an antiviral plant defense protein found in the root tuber of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor (prepro) protein, containing a 23 amino acid amino-terminal sequence (N) and a 20 amino acid carboxy-terminal extension (C). Various constructs of the TCS gene were expressed in transgenic tobacco plants and suspension cell culture to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression, localization, and toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble proteins (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the cauliflower mosaic virus 35S RNA promoter for constitutive expression. One hundred-fold lower expression levels were evident with gene constructs without the C-terminal coding sequences. Furthermore, severe phenotypic aberrations were apparent in the tobacco plants expressing the TCS gene constructs without the C-terminal coding sequence. In transgenic suspension cultures, rTCS was secreted at levels of 1--2 mg/L from callus carrying the modified prepro-TCS gene. In protoplast cultures, the levels of secreted rTCS increased to 6 mg/L. These results represent the highest reported expression levels of recombinant RIPs in transgenic plants to date. Recombinant trichosanthin (rTCS) was localized to the cell wall matrix of transgenic tobacco when the gene was expressed with the amino-terminal TCS sequence. The TCS amino-terminal sequence was also sufficient to direct localization of recombinant green fluorescent protein (rGFP), an intracellular protein, to the extracellular space of tobacco. Extracellular localization of the rTCS was confirmed via confocal microscopy, vacuum infiltration analysis, and protoplast and transgenic suspension culture studies.;The expressed trichosanthin was partially purified from total plant soluble protein extracts, using a five phase purification scheme. Approximately 63% of the rTCS was recovered during the entire purification process. Correct amino terminal processing of the rTCS, derived from the native prepro gene sequence, was confirmed by sequencing. The purified rTCS inhibited rabbit reticulocyte lysate protein translation similar to native TCS from T. kirilowii. Transgenic tobacco plants expressing the prepro rTCS exhibited enhanced resistance to systemic infection and local lesion development from cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) infections.