Purification, Molecular Cloning And Investigation On The Activity Mechanism Of Curcin From The Seeds Of Jatropha Curcas

Jatropha curcas L., a member of the family Euphorbiaceae, is widespread in tropical and subtropical countries. The toxicity of the whole seed of the species has been known for a long time, rendering them potentially toxic to seep, goat, rat, mice and human being, especially to children. The toxicity of the seed has been attributed in part to their irritating oil and in part to a protein component. A toxic protein, designated as curcin, was isolated and purified from J. curcas seeds by precipitation with 60% saturation of (NH^SC^ followed by chromatography on Sephedex G-100. The molecular weight of 28.2 kDa was determined by SDS-PAGE and the pi of 8.54 was determined by IEF-PAGE. The total neutral-surge content of purified curcin expressed, as mannose was 4.91%. Curcin exhibited a characteristic circular dichorism(CD) spectrum, a negative band centered at 217nm. It was estimated that curcin contained about 22.3% a-Helix, 43.5% |}-sheet and 34.2% random coil. N-terminal amino acids were A-G/Y-S/K-T/A-P/D-T-L-T-I-T-Y-D-A-T/A-A-D-K-K-N-Y-A-Q-F-I-K-D-L-R-E-A-F/A-G When the content of curcin applied is biger than 7.8 mg/L, it can agglutinate rabbit red cell. The antibody was detected by techniques of ELIST and Western blotting.Curcin strongly inhibits protein synthesis in rabbit reticulocyte lysate system with an ICso value of about 0.19?.011nmol/L. When rRNAs were simultanrous treated with curcin and aniline, a characteristic fragment of approximant 450 bp was obtained on 3.5% urea PAGE gel, which reveals that curcin has the activity of RNA N-glycosidase indeed. MTT assay was performed to determine the inhibitoryefficiently of curcin on SGC-7901, Sp2/0, Human hepatoma, HeLa and MRC. The values of ICsoof curcin on SGC-7901, Sp2/0 and Human hepatoma were (0.23 ± 0.004) mg/L, (0.66±0.015) mg/L, (3.16±0.031) mg/L respectively, but the protein is nontoxic to HeLa and MRC. Under the Electromagnetic Pulsed Fields (EMPF), the survival rate of HeLa cells were sharply decreased and apparent apoptotic cells were observed under microscope after treated with curcin. Meanwhile, apoptotic peak of sub-diploid was also observed in the Gl prophase of cell cycle. No regular ladder bands of cleaved DNA were observed on electrophoresis gel. The oral and intraperitoneal injection of IDso of curcin was found to be 104.1'37±29.447 mg/kg and 67.20 ± 10.445 mg/kg body weight of mice.Degenerate primers were designed in accordance with the N-terminal partial sequence of purified curcin. The full-length cDNA was cloned by RT-PCR and 5'-RACE, and sequenced (GenBank AY069946). The deduced amino acid sequence indicates that a protein precursor with 293 amino acid residues is first translated and then processed to a mature protein with 251 amino acids. The deduced amino acids share homology of 33%-56% with type I RIPs and A chain of type II RIPs. A genomic clone that specifies a single polypeptide precursor for curcin was isolated and sequenced using genomic walker technology (GenBank AF 469003). Nucleotide sequence analysis indicates that the gene contained an encoding frame and a 36 bp upstream before the first methionine codon. One putative TATA boxes and two possible CAAT box lie in the 5'-flanking region. No intron was found, which is typical of other RIPs genes that have been sequenced.pQE-Jl and pQE-J2 expression plasmids were constructed by inserting the FL-2 and CB1 DNA fragment into vector pQE-30, and the pQE-30-FL2 and pQE-30-CBl vectors were transferred in E. coil strain Ml5 (pREP4). Induced with Immol/L IPTG, the expressed product was identified by SDS/PAGE and Western blotting. After the recombinant protein was purified using Ni-NTA affinity column, it was found that the recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate system with an IC50 value of about 0.32±0.038nmol/L (CB1) and 0.44.±0.016nmol/L (FL2).