Ribosome‐Inactivating Protein Production from Trichosanthes kirilowii Plant Cell Cultures

Two ribosome‐inactivating proteins (RIPs) found in Trichosanthes kirilowii root tuber, trichosanthin (27 kDa) and TAP‐29 (29 kDa), have been reported to exhibit antiviral (including HIV‐1) and antitumor activities. Using SDS‐PAGE and Western blotting analyses, we have studied the production of intracellular and extracellular proteins from T. kirilowii callus grown on semisolid medium, callus grown in suspension, and Agrobacterium rhizogenes A4 transformed callus grown in suspension. This transformation resulted in callus rather than hairy root growth, although the growth rate of the transformed callus on hormone‐free medium was similar to that obtained for the nontransformed callus on hormone medium. Trichosanthin, identified through SDS/ PAGE and Western blotting, was detected only in root tuber and cell extracts of the transformed cell line. A 29‐kDa protein was found in intracellular extracts and extracellular solutions from all of the above samples; however, the highest yield was obtained from the broth of the agrobacterium‐transformed callus. Following ion‐exchange purification of the shake flask broth on a strong cation‐exchange column (S‐Sepharose), elution fractions containing the 29‐kDa protein showed a high degree of RIP activity, as evidenced by total inhibition of protein synthesis using an in vitro protein translation assay. The yield of the 29‐kDa protein recovered from the broth was greater than 1.0 % (w/w) of the dry weight of the callus. For comparison, the yield of TAP‐29 obtained by extraction of dried root tuber is on the order of 0.01% (w/w) of the dry weight (Lee‐Huang et al. , 1991); our estimate of the 29‐kDa protein from our fresh root tuber is between 0.4% and 1% (w/w) on a dry weight basis.