Expression of ricin A chain in <i>Escherichia coli</i>

O'Hare, Mary C.; Roberts, Lynne M.; Thorpe, Philip E.; Watson, Graham J.; Prior, Bernadette; Lord, J. Michael

doi:10.1016/0014-5793(87)80759-7

Cited by 91 publications

(41 citation statements)

References 15 publications

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“…Initial efforts to express high levels of RTA in E. coli were complicated by temperature-dependent aggregation phenomena (29,30). RTA was stabilized in a previous study by fusion to P-galactosidase (25) and in the present study by fusion to glutathione-S-transferase.…”

Section: Discussionmentioning

confidence: 57%

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

Tagge¹,

Chandler²,

Tang³

et al. 1996

J Histochem Cytochem.

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DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid.After transformation of JMi05 E. coli cells and induction with isopropylthio-~-galactoside (PIG), fusion proteins were bound to an immobilized glutathione matrix and r e " -binant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with icities were measured on seven cell lines for each A-chain and heterodimer. Although KDEL sequences enhanced cyplant rich B chain (RTB) similarly to plant RTA. Cell Cytotoxtotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin Cytotoxicity, Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in semitivity to KDELcontaining toxins correlated with altered &-tribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments. (J Histochem Cytochem 4Xl59-165, 19%)

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Section: Discussionmentioning

confidence: 57%

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

Tagge¹,

Chandler²,

Tang³

et al. 1996

J Histochem Cytochem.

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“…Previous attempts to express soluble, active RTA in E. coli have been complicated by temperature-dependent aggregation phenomena as well as minor alterations in RTA sequence (23,26). We have shown that RTA can be stabilized by fusion to P-galactosidase as described by Germino et al (7,8) and that soluble fusion protein is produced in high yield even in cultures grown at 37°C.…”

Section: Discussionmentioning

confidence: 99%

“…The yeast expression system (6) yields very small amounts of RTA, which is difficult to purify from total yeast extracts. Available bacterial expression systems (23,26) produce material that is cumbersome to purify and may have solubility problems, especially at temperatures of 37°C or higher.…”

mentioning

confidence: 99%

Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

et al. 1989

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The gene for the A chain of ricin toxin was fused to a j8-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.Ricin is a polypeptide toxin found in the seeds of the Ricinus communis (castor bean) plant. This 65-kilodalton (kDa) heterodimeric protein consists of a 32-kDa A chain (RTA) linked by disulfide bond to a 33-kDa B chain (RTB) (24,28). RTB is a lectin that binds to ,3-galactoside-terminated oligosaccharides on the surface of mammalian cells. After binding, the toxin is internalized and transported to an intracellular compartment (thought to be the trans-Golgi [25,30]), where the chains separate and RTA translocates across the vesicle membrane to the cytosol. In the cytosol, RTA catalytically inactivates protein synthesis by hydrolysis of the N-glycosidic bond of adenosine 4324 in the 28S RNA of the eucaryotic 60S ribosomal subunit (5). This depurination irreversibly inactivates the ribosome, possibly by altering the binding site for elongation factors (20). Under optimal conditions, a single molecule of RTA can inactivate 1,500 ribosomes per min, and a single molecule of RTA in the cytosol is sufficient to cause cell death (4).The three-dimensional structure of ricin determined by X-ray crystallography (21) showed a prominent deep cleft that was proposed as the enzyme-active site (21,27,28).

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“…Cloning and Expression of Recombinant Toxins-The vector pRICA, designed for expression of active RTA in the cytoplasm of Escherichia coli, has been described elsewhere (7). The RTA coding region with an additional 26 base pairs at the 5Ј-end is contained in an 873-base pair BamHI fragment.…”

Section: Methodsmentioning

confidence: 99%

Self-potentiation of Ligand-Toxin Conjugates Containing Ricin A Chain Fused with Viral Structures

Chignola¹,

Anselmi²,

Serra

et al. 1995

Journal of Biological Chemistry

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A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10 -20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90 -240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.Cell-surface structures mediating the efficient internalization of cell-bound molecules are frequently selected as targets of monoclonal antibody/ligand-toxin conjugates (immunotoxins (IT) 1 ) (1). Rapid internalization, however, is not always synonymous with fast intoxication rates of the target cells as a result of cell mechanisms leading to inactivation of the internalized IT molecules (e.g. recycling, degradation, slow routing to subcellular compartments competent for toxin translocation) (1). The ricin toxin A chain (RTA) is a potent ribosome-inactivating enzyme used in the synthesis of highly selective IT. However, RTA-based IT exert their effect at relatively high concentrations due to poor translocation of RTA to the cell cytosol from the endocytic compartments where the IT are internalized (1).Viruses utilize specialized envelope structures that allow them to enter the cytosol of the infected cells. We reasoned that it might be possible to modify a cytotoxic enzyme (i.e. RTA) by fusing it to a protein structure derived from viral envelopes, thus conferring to the cytotoxic enzyme the cytosol targeting properties of the virus. A peptide representing the primary sequence of the 25 N-terminal amino acids of protein G of the vesicular stomatitis virus envelope (KFT25) was found to have pH-dependent membrane destabilizing properties (2, 3). In particular, at low pH, KFT25 was shown to be hemolytic, to mediate hemagglutination, to be cytotoxic for mammalian cells, and to effect gross changes in cell permeability (2, 3). Such a virus-derived structure might be endowed with the ability to facilitate the translocation of heterologous proteins across cell membranes when they are routed to acidic intracellular compartments.The transferrin receptor is a cell-surface structure known to de...

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Expression of ricin A chain in Escherichia coli

Cited by 91 publications

References 15 publications

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

Self-potentiation of Ligand-Toxin Conjugates Containing Ricin A Chain Fused with Viral Structures

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