Purification, Cloning And Expression Of Ribosome Inactivating Protein From Gynostemma Pentaphyllum

Many Cucurbitaceae plants were known TO he Chinese medicine herbs and contain ribosome-inactivating proteins (RIP), a group of toxic proteins that have been proposed and demonstrated to play potential use as toxins in the therapy of a variety of human tumors and viruses including HIV. And much interest has been devoted to construct RIP transgenic crops for the protection from pathogen infection. RIP is a multi-activity protein, and different RIPs exhibit unique functions. Thus, a rapid screening method for novel RIP genes is in urgent need. Gynosiemma pentaphyllum, a member of Cucurbitaceae family, is a kind of herb with great value of Chinese medicine and widely manufactured for health care in China. However, there were no reports on active proteins from Gpentaphyllum.A novel RIP named Gynostemmin (27 kDa) was purified from the leaves and stems of Gpentaphyllum. It showed excellent anti-TMV activity. And the N-terminal 19 amino acid residues are 'DINFSLAGADGQTYN TFIA' (accession number P83206 in SWISS-PROT). which shared 10%~73% identities to other RIPs from plants and 37%~73% to other RJPs from Cucurbitaceae.Six novel RIP gene fragments (408'bp), 1 from Benincasa hispida and 5 from Cucurbit a moschaia. were isolated from Cucurbitaceae by applying a PCR strategy termed 'conserved regions cloning" using a pair of two degenerated primers deduced from the two blocks with strong amino acid conservation locating at the position 66-73 and 195-202 referring to the mature TCS sequence. Compared with the corresponding regions of 19 RIPs from Cucurbitaceae. 29 completely conserved amino acid residues were revealed, and 7 of them were aiso identical within other type I and type II RIPs from other families, including the proposed active amino acid residues (Gluiro. Arg163, and W192 in TCS).The partial coding region of Gynostemmin (609 bp, accession number AY075115 in GenBank) was cloned from the genomic DNA by using a pair of degenerated primers based on its N-terminal amino acid sequence and the highly conserved amino acid residues in the downstream of most RIPs fromCucurbitaceae. 5'RACE extended 33 bp upstream and revealed a signal peptide with 23 amino acid residues: MRVARFCTVVAILLYFGFHiAEC. Five cDNA sequences with 3'UTR were identified by 3 "RACE, the secondary structures of 3'UTR between GynostemminI and GynostemminII- V were remarkably different. And another 4 cDNA and 2 DNA sequences were obtained. They shared high homologies both at nucleotide acid ieve! and amino acid level. According to the 6 bp or 87 bp deletion, the RIP muitigene family from Gpemaphyllum could be divided into four groups. Group 1, including Gynostemmin [I, GP609. and CX8405A. with the 6 bp deletion at the position of 535-540. encoding 275 aa. Group 2. including DNA-750 and CX820, with the 87 bp deletion at the position of 122-208, encoding 248 aa. Group 3, including 5RACE and DNA-8001. without the 87 bp deletion, and the 6 bp deletion were uncertain because of the incomplete sequence. And group 4, including GynostemminI, III~V, EMUZW, and RHXJB. without the deletion of both 87 bp and 6 bp, encoding 277 aa.Bioinformatic analysis were performed to prepro-Gynostemminl on the base composition, codon preference, amino acid composition, pi value, molecular weight, secondary structure, prosite scan, prediction of protein localization sites. blast thtf Arabidopsis thaliana genome, and 3D structure modeling.GST-fusion and native RIP from Gp&nlaphyUum were expressed in E.coli by inserting the GP609 and RHXJB into the pGEX-2T vector, and DNA-8001 into the pET-5a vector, respectively. The native expressed product from E.coli BL21(DE3) strain, however, showed weak activity against TMV.GP609 and ZWEMU were inserted into pEmu-mcs-N, an expression vectorfor monocotyledon. And RHXJB was inserted into pKYLX71:35S2. a suitableexpression vector for dicotyledon, then introduced into Agrobacterium LBA4404 by triparental mating. Transgenic tobacco K-326 infected by the recombinant Agrobacterium was demonstrated by PCR. sequencing, and N...