1.Expression, Purification And Its Bioactivity Of Recombinant Ricin A Chain 2.Expression And Its Analgesic Activity Of Conotoxin M â…¦ A Fused With Glutathion S-transferase

Ricin toxin (RT) is a plant toxin isolated from the seeds of Ricinus Communis. It is one of the most powerful plant toxin discovered. The toxin protein consists of two chains linked by a disulfide bond. Ricin A chain (RTA) is an active chain and has specific N-glycosidase activity, which excises a specific adenine residue (A4324 in rat) from a highly conserved loop of 26 or 28S rRNA in 60S ribosomal subunits. This cleavage of adenine can lead to the disruption of ribosomal function, thereby, inhibits the protein synthesis and then cause the death of cells. Ricin B chain (RTB) can specifically recognize receptors on the cell membrane, which have a terminal galactose. RTB is responsible for the binding to the cell receptors and may facilitate the translocation of RTA. Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular compartments. Ricin toxin has wide-range anti-tumor spectrum. However, because RTA has no selectivity, it can damge normal cells too. These years, ricin and it's A chain (RTA) have been used to conjugate with monoclonal antibody to prepare immunotoxins(ITs). Immunotoxins have been used for cancer-targeted therapy, and many of them now are on clinical trials. Because expressed in Escherichia coli, ricin A chain is not degraded easily for its non-glycosylation. Our experiments lay a foundation for the researches that RTA conjugates a monoclonal antibody to synthesize immunotoxins and the immunotoxins are used for cancer-targeted therapy.Our goal is to express the ricin A chain (RTA) and its modification RTA-KDEL (ER-retrival signal) in Escherichia coli. In previous studies, we reported construction of expression vectors of RTA and its mutants. Briefly, the DNA sequence ofRTA-KDEL was created by polymerase chain reaction by using the original plasmid pGEM3zf(-)-RTA. The PCR products were directly cloned into the pT7Blue T vector and were then subjected to DNA sequencing to verify the desired sequence. The fragments digested with restriction endonucleases EcoR I and Hind III were subsequently cloned into the vector pKK223.3. E. coli JM 109 transformed with the expression plasmid was grown in M9 media to an optical density of 0.7. Expression was induced by adding IPTG to a final concentration of ImM and incubation with shaking at 30 for 3 h. In order to evaluate the effect of temperature on expression of rRTAs, we have investigated the distribution of the recombinant proteins expressed at different temperatures. To identify an optimal length of the incubation, the bacteria were induced at 30 for 1 h, 3 h and 5 h respectively. The distribution of the recombinant proteins was analyzed on 12% SDS-PAGE. After induced by 0.1 M IPTG, the pellet was resuspended in phosphate-buffered saline (PBS)/5 mM EDTA, and sonicated three times for 20 s at maximal output. Debris was removed by centrifugation at 15000 rpm for 15 min at 4. The supernatants were dialyzed against 50 mM phosphate buffer (PB) (pH 6.5), and recombinant RTAs (rRTAs) were affmitively purified on a Blue-Sepharose 6B column by elution with 0.65 M sodium chloride. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the purity. The fractions containing the rRTAs were collected, and the protein concentration was measured by Bradford method. The toxicities of RTA and its mutant RTA-KDEL were evaluated by the MTT assay in HeLa, MCF and ECV-304 cells following fluid-phase endocytosis.From our experiments, we have demonstrated: (1) the recombinant expression vectors of RTA and its mutants RTA-KDEL have been successfully constructed by PCR. (2) Expression of rRTA and rRTA-KDEL was induced with 0.1 mM IPTG at 30 for 3 h. SDS-PAGE showed that the target proteins with approximate molecular weight, 31 kD could be detected in the lysates of the bacteria. (3) The rRTA induced at 30 for 5h was soluble and fully active. In comparison to rRTA, a major part of rRTA-KDEL was expressed in a soluble style at 30 for 3h, whereas that expressed at 37 was insoluble. (4) The...