Isolation,Purification And Characterization Of Two Novel Lower Molecular Weight Ribosome-inactivating Proteins From The Seeds Of Cucurbitaceae Plants

This thesis was focused on the studies on isolation~, purification and characterization of two novel small ribosome-inactivating proteins ? trichokirin and Luffin S2 from Cucurbitaceae plants. 1 Isolation ... purification and characterization of a novel small ribosome-inactivating protein named S-trichokirin from the seeds of Trichosanthes kirilowii A new small ribosome-inactivating protein named S-trichokirin from the seeds of Trichosanthes kirilowii was obtained by ammonia sulfate precipitation, CM-52 ion exchange chromatography, Sephacryl 5-100 gel filtration and FPLC Mono S ion exchange chromatography. The purity of S-trichokirin was also demonstrated by reverse-phase HPLC (RP-HPLC) and mass spectrometer. Its yield was only about 0.4 mg per defatted seeds powder. S-trichokirin has molecular weight about 8 kD, as determined by 15% SDS-PAGE and 15% Tris-Tricine PAGE. It was proved to be a strong basic protein with p1 about pH 9.5 by 13.5% acidic PAGE. The reaction mechanism of S-trichokirin is the same as that of trichosanthin, which is an RNA N-glycosidase. It had a strong inhibitory effect on protein biosynthesis in rabbit reticulocyte lysate with IC50 of 1.15 X I 0?mol/L. 5- trichokirin may be used as a new efficient moiety of immunotoxin. 2 Isolation% purification and characterization of a new low molecular weight ribosome-inactivating protein桳uffin S2 from the seeds of Luffa cylindrica Luffin S2 was another small ribosome-inactivating protein originated from Luffa cylindrica seeds. Luffin ~2 could be obtained by ammonia 122 ~EE~F~ sulfate precipitation, CM-52 ion exchange chromatography, Sephadex G- 25 desalting and FPLC Mono S ion exchange chromatography. Its yield was about 1.5 mg per lOOg dry seeds powder. With the same mechanism inactivating ribosomal RNA, Luffin ~2 exhibited a strong inhibitory effect on protein biosynthesis in rabbit reticulocyte lysate (IC50: 1.50 X 10 ?mol/L). On the basis of immunizing Wister rats with Luffin S2 purified after FPLC Mono-S , both polyclonai and monocional antibodies were prepared simultaneously. Although polyclonal anti- Luffin S2 serum had high titers, but their specificities were very poor. The monoclonai anti-Luffin S2 antibodies showed high specificity and could be used to detect nature Luffin ~2 by ELISA or Western-blotting. The nine amino acids of N-terminal of Luffin S2 was Pro Arg Arg Giy Gin ?Glu Ala ?Phe Asp determined by Protein Sequence . Then , partial eDNA of Luffin ~2 was cloned by RT- PCR with two primers ( one was degenerate primer for 5?terminal primer and the other was a general primer containing oligo-dT). The cloned cDNA of Luffin ~2 was composed by 198 bp coding sequnece and 3?terminal 87 bp noncoding sequence. Luffin S2 had molecular weight about 7.85 kD concluded by its cDNA, which was identical to the result determined by 15% SDS-PAGE. A recombinant plasmid pET28a-c(+)S2 was constructed containing Luffin ~2 gene and expressed in Escherichia coil. Recombinant Luffin S2 could be expressed in a very low yield that it must be detected by ELISA, but the crude product purified by Ni-NTA affinity-column exhibited obvious RNA N-glycosidic activity on rat liver rRNA. The low-level...