Study Of Small Ribosome-inactivating Proteins (Peptides) From The Seeds Of Luffa Cylindrica And Trichosanthes Kirilowii

Small ribosome-inactivating proteins were a type of low molecular weight (8-14 kD)proteins which can inhibit protein biosynthesis. They all have N-glycosidaseactivities. Small ribosome-inactivating proteins may have potential for new toxinmoiety for construction of immunotoxins. And they may have the advantage inreducing the immunogenicity of immunotoxin due to their small molecules. Thisdissertation was focused on the studies on isolation, purification, characterization,cDNA cloning and expression of three small ribosome-inactivating proteins fromcucurbitaceae plants.A peptide designated Luffin P1 with molecular weight of 5226.1 Da was purifiedfrom the seeds of Luffa cylindrica by ammonia sulfate precipitation, CM-52 ionexchange chromatography, and FPLC Mono S ion exchange chromatography. Thesequence of N-terminal 11 amino acids of Luffin P1 was identical with the partialN-terminal sequence (from G3 to R13) of 6.5K Arg/Glutamate rich peptide, whichwas also isolated from the seeds of Luffa cylindrica. Besides, Luffin P1 has a veryhigh homology with a trypsin inhibitor, named C2 peptide, from pumpkin seeds.Interestingly, the purified Luffin P1 not only showed a strong inhibitory activity onprotein synthesis in rabbit reticulocyte lysate system with IC50 of 0.88 nmol/L, butalso had an obvious trypsin inhibitory activity with IC50 of 22 μmol/L. Besides, thegel filtration results of Luffin P1 implied the existence of polymer form of Luffin P1and the polymers might be formed by electrostatic interactions among monomers.Furthermore, the analyzed results of N-glycosidase activity in the different solutions iiiivsuggested that the polymerization of Luffin P1 enhanced its activity and polymerform of Luffin P1 might be the active form of enzyme. Luffin P1 was the smallestpeptide yet report that has translational inhibitory activity. Its cDNA had been clonedby RT-PCR. The results of comparison on sequence homology suggested that LuffinP1 may be derived from a vicilin like precursor which is a storage protein in theseeds of Luffa cylindrica.The cDNA of Luffin S2, which was also a small ribosome-inactivating protein fromthe seeds of Luffa cylindrica, was cloned by 3' RACE. And a recombinant plasmid ofGST and Luffin S2 fusion gene pGEX-4T-S2 was constructed and expressed inEscherichia coli successfully. About 40 mg purified soluble recombinant fusionprotein GST-Luffin S2 can be obtained from 1 liter bacteria cultures. The fusionprotein showed strong rRNA N-glycosidase activity. It is the first smallribosome-inactivating protein produced by gene-engineering methods.A novel peptide from the seeds of Trichosanthes kirilowii, trichokirin-S1, waspurified by extraction of protein body, ammonia sulfate precipitation, Blue-gelaffinity chromatography, FPLC Mono S ion exchange chromatography andSuperose12 gel filtration chromatography. Its molecular weight was determined to be11426 Da by MALDI-TOF MS analysis. Its reaction mechanism to inactive ribosomeis the same as that of trichosanthin, a single chain ribosome-inactivating protein,which is an rRNA N-glycosidase. The purified trichokirin-S1 showed a stronginhibitory activity on protein synthesis in cell-free rabbit reticulocyte lysate system,with IC50 of 0.71 nmol/L. A strain of hybridoma which can secrete monoclonalantibody against trichokirin-S1 was obtained by cell-fusion technique. The antibodycan react with trichokirin-S1 specifically and was successfully used to detecttrichokirin-S1 by Western-blotting test.END-END ■ Key words: ribosome-inactivating protein, N-glycosidase, cDNA cloning, geneexpression, fusion protein GST-Luffin S2...