1.The Expression And Purification Of Ricin A Chain And Its Cytotoxicity 2.Preparation Of Monoclonal Antibody Against Ricin A Chain

[Backgrounds]Many plants produce toxic proteins known as ribosome inactivating proteins (RIPs) whose functions mainly serve as a defensive tool against invasion of other organisms. Ricin is a heterodimeric RIP comprised of A and B chains joined through a disulfide bond. It is toxic for most mammalian cells since ricin A chain (RTA) is an RNA specific N-glycosidase that removes a specific adenine residue in a highly conservative region from among over 4000 nucloside residues present in 28S rRNA, and causes protein synthesis inhibition and cell death. A single molecule of RTA is able to depurinate 1500-2000 ribosomes per minute. Ricin B chain (RTB) is a galactose-specific lectin which binds to the receptors on cell surfaces, and may enhance RTA translocation by forming a pore in the membrane of intracellular organelles.Ricin enters the cells by receptor-mediated endocytosis. In order to reach the substrate (ribosomes), RTA must translocate into the cytosol. Translocation from endocytic compartments to the cytosol is the essential and rate-limiting step in the intoxication process of most toxins such as ricin, Diphtheria toxin, Shiga toxin and Pseudomonas exotoxin (PE). A number of these toxins are transported to trans-Golgi network (TGN), and in many cases such transport to the TGN is required for the translocation and cytotoxicity. In deed, 5% of the ricin endocytosed by cells has been shown to reach the TGN. It is known from studies using brefeldin A (BFA) that disruption of TGN can block the cytotoxicity of ricin, suggesting that the intracellular compartment may be an important part of the uptake pathway.Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular organelles. In this study, a trans-Golgi retention peptide signal YQRL was fused to the C-terminus of ricin A chain (RTA) by polymerasechain reaction. The recombinant RTA and RTA-YQRL were expressed in Escherichia coli using plasmid pKK223.3 under the control of a tac promoter. The recombinant proteins were purified by affinity chromatography on a Blue-Sepharose 6B column. The cytotoxicities of RTA and the fusion toxin RTA-YQRL were measured by the MTT assay in HeLa, SKOV-3, and WISH cells following fluid-phase endocytosis.[Methods]1. Expression and purification of recombinant RTAsThe expression plasmid Pkk223.3-RTA was introduced into E. coli JM 109 by CaCl2-mediated method. The cells were grown until an optical density at 600 nm reached 0.6. Expression was induced in the presence of IPTG (final concentration 1 mM) and incubated at30â—‹ for 3h). Cells were then harvested by centrifugation and the pellet was resuspended in phosphate-buffered saline (PBS) containing 5 mM EDTA. After sonication, debris was removed by centrifugation at 10000 x g for 15 min at 4â—‹. The supernatants were dialyzed against 50 mM phosphate buffer (PB) (pH 6.5), and passed through a Blue-Sepharose 6B column (3x30 cm). The bound rRTA and rRTA-YQRL were eluted with 0.65 M sodium chloride. 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to analyze the purity and the protein concentration was measured by the Bradford method.2. Cytotoxicity assayHela, SKOV-3, and WISH cells were plated out in 96-well plates at a density of 1.0x104 cells per well in the serum-free RPMI-1640 medium. Various concentrations of rRTA and rRTA-YQRL were added into the wells and incubated for 6 h to allow internalization of toxins by fluid-phase endocytosis. The cells were then incubated in RPMI-1640 medium containing 10% fetal calf serum for another 24 h. Cell viability was measured by MTT assay. In this assay, a tetrazolium salt (MTT) is converted to a blue formazan product by enzymes active only in living cells. The amount of formazan produced can be quantitated using a microtitre plate reader. In order to get precise data, each concentration was set for 5 wells.[Results]1. Expression of recombinant RTA and RTA-YQRLE. coli JM 109 transformed with the expression plasmid was grown and i...