Selection and characterization of peptide memitopes binding to ricin

A combinatorial random peptide display library expressed in E. coli was employed to identify short, linear peptide sequences that showed affinity for ricin and could be used as reagents for detection and identification of ricin. One peptide, P3, from a collection of four short peptides showed specific binding to ricin. The kinetic analysis of this peptide binding to the ricin showed lower equilibrium binding constants for the peptide P3 than monoclonal antibody. This is attributed due to both slower association and faster dissociation rates for the peptide P3. The random ricin peptide P3 binds to ricin with a K_D of 1 M versus the antibody's K_D of 14 nM. This particular peptide memitope P3 against ricin showed specific binding to ricin without any significant cross-reactivity against other proteins such as bovine serum albumin (BSA), lysozyme and natural bacterial toxins such as Staphylococcal enterotoxins A and B. The results provided proof-of-principal that peptide memitopes are another choice of reagents due to ease in production to be used for the detection of highly toxic bio-threat or biowarfare agents such as ricin.     

Structure of ricin B-chain at 2.5 A resolution

The heterodimeric plant toxin ricin has been refined to 2.5 A resolution. The B-chain lectin (RTB) is described in detail. The protein has two major domains, each of which has a galactose binding site. RTB has no regular secondary structure but displays several omega loops. Each RTB domain is made of three copies of a primitive 40 residue folding unit, which pack around a pseudo threefold axis. In each domain, galactose binds in a shallow cleft formed by a three residue peptide kink on the bottom and an aromatic ring on the top. At the back of the cleft, an aspartate forms hydrogen bonds to the C3 and C4 hydroxyls of galactose, whereas a glutamine bonds to the C4 alcohol, helping to define specific epimer binding. In addition to analyzing the sugar binding mechanism, the assembly of subdomain units around the pseudo threefold axis of each domain is described. The subdomains contribute conserved Trp, Leu, and Ile residues to a compact central hydrophobic core. This tight threefold binding probably drives the peptide folding and stabilizes the protein structure.     

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fussion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.     

Expression of functional hexahistidine-tagged ricin B in tobacco

Ricin B (RTB), the lectin subunit of ricin, shows promise as an effective mucosal adjuvant and carrier for use in humans. In order to obtain a recombinant plant source of RTB that is devoid of the toxic ricin A subunit, we expressed RTB in Nicotiana tabacum. RTB was engineered with an N-terminal hexahistidine tag (His-RTB), which may affect protein stability. Lactose-affinity purification of His-RTB from leaves yielded three major glycosylated products of 32, 33.5 and 35 kDa. Their identity as RTB was verified by mass spectrometry and immunoblotting with anti-ricin antibodies. Functionality of His-RTB was confirmed by binding to asialofetuin, lactose and galactose.     

Construction and Characterization of Versatile Cloning Vectors for Efficient Delivery of Native Foreign Proteins to the Periplasm ofEscherichia coli

Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones inEscherichia coliinhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic forE. coli,but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit ofE. coliheat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield inE. coli.Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm ofE. coli.We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, usingphoAfromE. coli,we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using thepspAgene that encodes pneumococcal surface protein A fromStreptococcus pneumoniae,we produced a 299-residue amino-terminal fragment of PspA inE. coliin large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure–function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm inE. coli.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

Immunotoxins up to the present day

Immunotoxins consist of monoclonal or polyclonal antibodies conjugated to bacterial or plant toxins. The toxins used are typically of the A-B type in which a toxic A chain is coupled to a B chain responsible for cell binding and facilitation of A chain entry into the cytosol. Two broad strategies have been followed: coupling intact toxins, or A chains alone, to antibodies. This review examines current progress inin vitro andin vivo research, including recent clinical studies, concentrating principally on ricin or ricin A chain conjugates. The future role of conjugates using membrane-acting toxins, immunolysins, is also discussed.     

Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1

Summary The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.Abbreviations CMCase carboxymethylcellulase - celA gene coding for CeIA - CelA cellodextrinase - ORF open reading frame - phoA gene encoding alkaline phosphatase - pNPC p-nitrophenyl--d-cellobioside     

Binding of Lactose and Galactose to Native and Iodinated Ricin D

The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the low-affinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.

By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.

From these results, it was inferred that the binding site abolished by the iodination is the LA-binding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.     

Clearance of false-positive antigen-antibody reactions of a diagnostic antigen produced inEscherichia coli with human sera

Although many pharmaceutically useful proteins are produced inE. coli expression system, it is very rare for the system to be used in the production of diagnostic antigen due to a major problem,i.e., false-positive reaction ofE. coli host-derived proteins contaminating purified diagnostic antigen with human sera. The N (nucleocapsid) protein of Seoul virus causing haemorrhagic fever with renal syndrome (HFRS) was produced inE. coli BL21 (DE3), and used for the detection of N protein-specific antibodies in human sera. Using the N protein as a diagnostic antigen of HFRS, the false-positive reaction was cleared by merely mixing the test sera with the extract ofE. coli host strain not harboring expression plasmid.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

Purification,partial characterization,and subcellular localization of a 38 kilodalton,calcium-regulated protein of Rhizobium fredii USDA208

Calcium is essential for the growth of rhizobia and the formation of nitrogen-fixing root-nodules on legumes, but its precise role in these processes remains unknown. We have found that Rhizobium fredii USDA208 accumulates a major 38 kDa protein when grown in media supplemented with 0.3–2 mM CaCl₂. We have purified this protein and raised polyclonal antibodies against it. The protein initially is synthesized as a 40 kDa precursor which subsequently undergoes calcium-dependent processing to give rise to the mature polypeptide. Subcellular and immunocytochemical localization studies indicate that the 38 kDa protein accumulates preferentially in the periplasmic space. Its N-terminal sequence, AETIKIGVAGPMTG, shows significant homology to the N-termini of amino acid binding proteins from the periplasm, including leucine-, isoleucine-, and valine-specific binding proteins of Pseudomonas aeruginosa and Escherichia coli and a leucine-specific binding protein of E. coli. The R. fredii protein does not, however, bind [³H]-leucine. The 38 kDa protein is encoded by the bacterial chromosome. It is absent in several rhizobia other than R. fredii, but antigenically related polypeptides are present in Escherichia coli and Erwinia carotovora subsp. carotovora.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Bacterial and plant enterotoxin B subunit-autoantigen fusion proteins suppress diabetes insulitis

Several bacterial and plant enterotoxin B subunit-islet autoantigen fusion proteins were compared for their ability to serve as islet autoantigen carriers and adjuvants for reduction of pancreatic islet inflammation associated with type 1 diabetes. The cholera toxin B subunit (CTB), the heat-labile toxin B subunit from enterotoxigenic Escherichia coli (LTB), the Shigella toxin B subunit (STB), and the plant toxin ricin B subunit (RTB) were genetically linked to the islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The adjuvant-autoantigen gene fusions were transferred to a bacterial expression vector and the corresponding fusion proteins synthesized in E. coli. The purified adjuvant-autoantigen proteins were fed to 5-wk-old nonobese diabetic (NOD) mice once a week for 4 wk. Histological examination of pancreatic islets isolated from inoculated mice showed significant levels of insulitis reduction in comparison with uninoculated mice. The ratio of serum anti-INS and anti-GAD IgG_2c to IgG₁ antibody isotype titers increased in all ligand-autoantigen inoculated animal groups, suggesting an increase in effector Th2 lymphocytes in B subunit-mediated insulitis suppression. The results of these experiments indicate that bacterial and plant enterotoxin B subunit ligand-autoantigens enhance insulitis reduction in NOD mice. This research prompts further exploration of a multiadjuvant/autoantigen co-delivery strategy that may facilitate type 1 diabetes prevention and suppression in animals and humans.     

Recognition and interaction of small rings with the ricin A-chain binding site

Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings. Proteins 31:33–41, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Summary From libraries of EcoRI fragments of Salmonella thyphimurium and Escherichia coli DNA in gt7, phages could be isolated that carry mglB, the structural gene of the galactose-binding protein as well as other mgl genes. Lysogenization of an E. coli mutant carrying a defective galactose-binding protein with gt7 mglB (Salmonella) restores full galactose transport and galactose chemotaxis. Both the E. coli mutant protein as well as the wild-type Salmonella galactose-binding protein are synthesized in this strain. The EcoR1 fragments of both organisms carrying the mgl genes were 6 Kb long. They were subcloned into the multicopy plasmid pACUC184. The hybrid plasmid containing the Salmonella mgl DNA gives rise to the synthesis of large amounts of galactose-binding protein in the periplasm of E. coli. The protein can be precipitated by antibodies against the E. coli binding protein and is identical to the fully processed protein isolated from Salmonella typhimurium LT2. In vitro protein synthesis (Zubay-system) with either gt7 mgl phages as well as the hybrid plasmid as DNA matrix produces the galactose-binding protein mainly in precursor form that is precipitable by specific antibodies.     

The Status and the Role of Glutathione under Disturbed Ionic Balance and pH Homeostasis in Escherichia coli

The study of glutathione status in aerobically grown Escherichia coli cultures showed that the total intracellular glutathione (GSH_in + GSSG_in) level falls by 63% in response to a rapid downshift in the extracellular pH from 6.5 to 5.5. The incubation of E. coli cells in the presence of 50 mM acetate or 10 g/ml gramicidin S decreased the total intracellular glutathione level by 50 and 25%, respectively. The fall in the total intracellular glutathione level was accompanied by a significant decrease in the (GSH_in : GSSG_in) ratio. The most profound effect on the extracellular glutathione level was exerted by gramicidin S, which augmented the total glutathione level by 1.8 times and the (GSH_out : GSSG_out) ratio by 2.1 times. The gramicidin S treatment and acetate stress inhibited the growth of mutant E. coli cells defective in glutathione synthesis 5 and 2 times more severely than the growth of the parent cells. The pH downshift and the exposure of E. coli cells to gramicidin S and 50 mM acetate enhanced the expression of the sodA gene coding for superoxide dismutase SodA.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.