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941.
Anti-Tac(Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interleukin 2 receptor bearing cells 总被引:8,自引:0,他引:8
J K Batra D FitzGerald M Gately V K Chaudhary I Pastan 《The Journal of biological chemistry》1990,265(25):15198-15202
Anti-Tac(Fv)-PE40 is a chimeric single chain immunotoxin in which anti-Tac variable heavy and light chains held together by a peptide linker are attached to PE40, a truncated form of Pseudomonas exotoxin. This molecule was shown to be extremely cytotoxic for interleukin 2 (IL2) receptor bearing cells in tissue culture (Chaudhary, V. K., Queen, C., Junghans, R. P., Waldmann, T. A., FitzGerald, D. J., and Pastan, I. (1989) Nature 339, 394-397). Here we describe various forms of anti-Tac(Fv)-PE40 protein in which the order of the variable domains of anti-Tac has been switched and also three different types of peptide linkers have been used. All these proteins were purified to near homogeneity and were found to have similar cytotoxic activities against various human cells expressing the p55 subunit of the IL2 receptor. Anti-Tac(Fv)-PE40 was also found to have a very potent suppressive activity against phytohemagglutinin-activated human lymphoblasts and in a human mixed lymphocyte reaction. Anti-Tac(Fv)-PE40 appeared in the blood rapidly in mice after intraperitoneal administration and could be detected in the blood for up to 8 h. Anti-Tac(Fv)-PE40 warrants evaluation as an anti-tumor and immunosuppressive agent in humans. 相似文献
942.
943.
Cell-specific toxicity of a chimeric protein composed of interleukin-6 and Pseudomonas exotoxin (IL6-PE40) on tumor cells.
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IL6-PE40 is a chimeric toxin composed of human interleukin-6 (IL6) linked by a peptide bond to PE40, a form of Pseudomonas exotoxin (PE) devoid of its cell recognition domain. To identify cancer cell lines with high numbers of IL6 receptors and to assess the usefulness of IL6-PE40 as a possible anticancer agent, we evaluated the toxicity of IL6-PE40 on a variety of tumor cell lines and demonstrated that certain human myeloma and hepatoma cell lines were particularly sensitive. IL6 binding to selected hepatoma and myeloma cell lines were determined by using [125I]IL6. IL6 receptor mRNA levels were measured by polymerase chain reactions. When comparisons were made among different hepatoma cell lines, the sensitivity to IL6-PE40 correlated with the number of IL6 receptors. However, the hepatoma line PLC/PRF/5, which contains 2,300 IL6 receptors, was more sensitive to IL6-PE40 (amount of protein required to inhibit protein synthesis by 50% was 5 ng/ml) than both the myeloma cell lines U266 and H929 (for both cell lines, the 50% inhibitory dose was 8 ng/ml), which contain 15,500 and 16,500 IL6 receptors, respectively. RNA analysis confirmed that the sensitivity of these cells to IL6-PE40 and the amount of IL6 receptor RNA detected did not correlate. These data suggest that factors in addition to the number of IL6-binding sites contribute to the sensitivity of cells to IL6-PE40. 相似文献
944.
A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. 总被引:22,自引:0,他引:22
Many proteins produced in Escherichia coli accumulate in inclusion bodies. We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial inclusion bodies containing a recombinant single-chain immunotoxin, B3(Fv)-PE38KDEL. This recombinant molecule is composed of the variable domains of monoclonal antibody B3 (B3(Fv)) fused to a truncated mutant form of Pseudomonas exotoxin A (PE38KDEL). This immunotoxin kills carcinoma cells in vitro, causes tumor regression in animal tumor models, and is being developed as an anti-cancer therapeutic agent (Brinkmann et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8616-8620). Like many other recombinant proteins, B3(Fv)-PE38KDEL is produced in E. coli in inclusion bodies and must be denatured and refolded to become active. This requires correct folding, formation of native disulfide bonds, and the association of different domains. All these steps are strongly dependent on the renaturation conditions used. Optimum conditions of refolding were obtained by the addition of reduced and oxidized thiol reagents to promote disulfide bond formation and the addition of a labilizing agent such as L-arginine. Furthermore, the necessity to reactivate proteins at low protein concentrations due to its tendency to aggregate at high concentrations was overcome by a step-by-step addition of denatured and reduced protein into the refolding solution. This approach should be useful for the production of active forms of other recombinant proteins. 相似文献
945.
S Kasturi A Kihara D FitzGerald I Pastan 《The Journal of biological chemistry》1992,267(32):23427-23433
Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency. 相似文献
946.
Redirecting Pseudomonas exotoxin 总被引:2,自引:0,他引:2
Pseudomonas exotoxin (PE) is a three-domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inactivating protein synthesis. The pathway of toxin entry includes binding to a surface receptor, internalization via coated pits and endosomes, proteolytic processing, reduction of disulfide bonds and finally the translocation of an enzymatically active C-terminal fragment to the cytosol. Once in the cytosol this fragment inhibits protein synthesis by ADP ribosylating elongation factor 2. Because of its potency PE and its derivatives have been directed to kill various target cells. It is hoped this strategy will lead to the development of a novel kind of therapeutic agent for the treatment of various human diseases including cancer, AIDS and various immunological disorders. 相似文献
947.
David C. Heimbrook Steven M. Stirdivant Janet D. Ahern Nancy L. Balishin Denis R. Patrick Gwynneth M. Edwards Deborah Defeo-Jones David J. FitzGerald Ira Pastan Allen Oliff 《Journal of industrial microbiology & biotechnology》1991,7(3):203-207
Summary Transforming growth factor-alpha (TGF)-pseudomonas exotoxin-40 (PE40) is a chimeric protein consisting of an N-terminal TGF domain fused to a C-terminal 40-kDa segment of the pseudomonas exotoxin A protein. TGF-PE40 exhibits the receptor binding activity of TGF and the cell killing activity of PE40. In the current study, we report that a modified TGF-PE40 derivative significantly prolongs the survival of nude mice bearing tumors derived from cell lines which express the epidermal growth factor receptor (EGFR). In addition, the therapeutic benefit of this protein is mediated by specific binding to the EGF receptor. These results indicate that a therapeutic window exists in vivo for the use of some growth factor-toxin fusion proteins as anticancer agents. 相似文献
948.
949.
950.
Ceth W. Parker Augusto S. Auler Michael D. Barton Ira D. Sasowsky John M. Senko 《Geomicrobiology journal》2018,35(4):311-322
The banded iron formations (BIF) of Brazil are composed of silica and Fe(III) oxide lamina, and are largely covered by a rock cap of BIF fragments in a goethite matrix (canga). Despite both BIF and canga being highly resistant to erosion and poorly soluble, >3,000 iron ore caves (IOCs) have formed at their interface. Fe(III) reducing microorganisms (FeRM) can reduce the Fe(III) oxides present in the BIF and canga, which could account for the observed speleogenesis. Here, we show that IOCs contain a variety of microbial taxa with member species capable of dissimilatory Fe(III) reduction, including the Chloroflexi, Acidobacteria and the Alpha- Beta- and Gammaproteobacteria; however, Fe(III) reducing enrichment cultures from IOCs indicate the predominance of Firmicutes and Enterobacteriaceae, despite varying the carbon/electron donor, Fe(III) type, and pH. We used model-based inference to evaluate multiple candidate hypotheses that accounted for the variation in medium chemistry and culture composition. Model selection indicated that none of the tested variables account for the dominance of the Firmicutes in these cultures. The addition of H2 to the headspace of the enrichment cultures enhanced Fe(III) reduction, while addition of N2 resulted in diminished Fe(III) reduction, indicating that these Enterobacteriaceae and Firmicutes were reducing Fe(III) during fermentative growth. These results suggest that fermentative reduction of Fe(III) may play a larger role in iron-rich environments than expected. Our findings also demonstrate that FeRM are present within the IOCs, and that their reductive dissolution of Fe(III) oxides, combined with mass transport of solubilized Fe(II) by groundwater, could contribute to IOC formation. 相似文献