RIBOSOME-INACTIVATING PROTEIN-PRODUCTION FROM TRICHOSANTHES KIRILOWIIPLANT-CELL CULTURES

Two ribosome-inactivating proteins (RIPs) found in Trichosanthes kiril owii root tuber, trichosanthin (27 kDa) and TAP-29 (29 kDa), have been reported to exhibit antiviral (including HIV-1) and antitumor activit ies. Using SDS-PAGE and Western blotting analyses, we have studied the production of intracellular and extracellular proteins from T. kirilo wii callus grown on semisolid medium, callus grown in suspension, and Agrobacterium rhizogenes A4 transformed callus grown in suspension. Th is transformation resulted in callus rather than hairy root growth, al though the growth rate of the transformed callus on hormone-free mediu m was similar to that obtained for the nontransformed callus on hormon e medium. Trichosanthin, identified through SDS/PAGE and Western blott ing, was detected only in root tuber and cell extracts of the transfor med cell line. A 29-kDa protein was found in intracellular extracts an d extracellular solutions from all of the above samples; however, the highest yield was obtained from the broth of the agrobacterium-transfo rmed callus. Following ion-exchange purification of the shake flask br oth on a strong cation-exchange column (S-Sepharose), elution fraction s containing the 29-kDa protein showed a high degree of RIP activity, as evidenced by total inhibition of protein synthesis using an in vitr o 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 c allus. For comparison, the yield of TAP-29 obtained by extraction of d ried 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.