Biosynthesis and bioactivity of trichosanthin in cultured crown gall tissues of Trichosanthes kirilowii Maximowicz.

Trichosanthin (TCS) from Trichosanthes kirilowii Maximowicz (T. kirilowii) can be used to treat choriocarcinoma. In this work, we established a novel system to produce TCS in crown gall tissues of T. kirilowii infected by Agrobacterium tumefaciens C58 (A. tumefaciens). In the crown gall tissues, a nopaline synthase (NOS) gene of A. tumefaciens was identified by polymerase chain reaction (PCR), and nopaline accumulation was confirmed by a high-voltage filter paper electrophoresis. Furthermore, we optimized conditions to culture the crown gall tissues able to grow fast and produce TCS in an auxin-free medium, and found that a fungal elicitor of Armillaria mellea was capable of stimulation of TCS secretion into the medium. Moreover, we identified that the TCS purified from the crown gall tissues could induce gastric cancer cell death. These data underscore the usefulness of our system as an inexpensive and virtually unlimited source of TCS.

[Prokaryotic expressed trichosanthin and other two proteins have anti-fungal activity in vitro].

DNAs encoding Trichosanthin, tobacco class I Chitinase and tobacco class I beta-1, 3-Glucanase were expressed in E. coli, respectively. The expression products were assayed for their anti-fungal activity. All of three kinds of proteins show anti-fungal activity. When two of them combined, this activity was enhanced greatly. When three of them combined, the stronger anti-fungal activity was observed.

Biosynthesis of defense-related proteins in transformed root cultures of Trichosanthes kirilowii Maxim. var japonicum (Kitam.).

We have established transformed ("hairy") root cultures from Trichosanthes kirilowii Maxim. var japonicum Kitam. (Cucurbitaceae) and four related species to study the biosynthesis of the ribosome-inactivating protein trichosanthin (TCN) and other root-specific defense-related plant proteins. Stable, fast-growing root clones were obtained for each species by infecting in vitro grown plantlets with Agrobacterium rhizogenes American Type Culture Collection strain 15834. Each species accumulated reproducibly a discrete protein pattern in the culture medium. Analysis of the extracellular proteins from T. kirilowii var japonicum root cultures showed differential protein accumulation in the medium during the time course of growth in batch cultures. Maximum protein accumulation, approaching 20 micrograms/mL, was observed at mid-exponential phase, followed by a degradation of a specific protein subset that coincided with the onset of stationary phase. Two major extracellular proteins and one intracellular protein, purified by ion-exchange and reverse-phase high-performance liquid chromatography, were identified as class III chitinases (EC 3.2.1.14) based on N-terminal amino acid sequence and amino acid composition homologies with other class III chitinases. The Trichosanthes chitinases also showed reactivity with a cucumber class III chitinase antiserum and chitinolytic activity in a glycol chitin gel assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that normal and transformed T. kirilowii var japonicum roots accumulated only low levels of TCN (approximately 0.5% total soluble protein). Storage roots from the plant displayed protein and antigen patterns different from root cultures and produced TCN as the dominant protein. Roots undergoing secondary growth and differentiation exhibited patterns similar to those of storage roots, including increased TCN levels, indicating that high production of TCN is associated with induction of secondary growth in roots.

Rapid, high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector.

alpha-Trichosanthin, a eukaryotic ribosome-inactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. The recombinant alpha-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.

High level synthesis of biologically active recombinant trichosanthin in Escherichia coli.

Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase-directed promoter. Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions. Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein. Its IC50 was approximately 0.13 nM in an in vitro rabbit reticulocyte translational system and a dose of around 35 micrograms protein per 25 g body weight was sufficient to induce complete abortion in mice. Form II rTCS had a propeptide of 19 aa at the C-terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.

Cloning of trichosanthin cDNA and its expression in Escherichia coli.

Several cDNA clones coding for trichosanthin (TCS) have been isolated from a cDNA library prepared from the poly(A)+RNA of the root tuber of Trichosanthes kirilowii Maximowicz. The nucleotide sequence codes for a protein of 289 amino acids (aa) including a putative signal peptide of 23 aa and an extra 19 aa at the C terminus; the latter two have not been found in TCS obtained from the natural product [Collins et al., J. Biol. Chem. 265 (1990) 8665-8669]. Recombinant TCS (reTCS) was synthesized in Escherichia coli, in which the cDNA without the signal sequence was expressed under the control of the trc promoter; reTCS was detected by a rabbit anti-TCS antiserum.