Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Ligand design for molecule-based magnetic and/or conducting materials

Work in the area of molecule-based magnetic and/or conducting materials is presented in two projects. The first project describes the use of 4,4’-bipyridine as a scaffold for the preparation of a new family of tetracarboxamide ligands. Four new ligands I-III have been prepared and characterized and the coordination chemistry of these ligands is presented. This project was then extended to exploit 4,4’-bipyridine as a covalent linker between two N3O2 macrocyles. In this respect, three dimeric macrocycles have been prepared IV-VI. Substitution of the labile axial ligands of the Co(II) complex IV by [Fe(CN)6]4- afforded the self-assembly of the 1-D polymeric chain {[Co(N3O2)H2O]2Fe(CN)6}n•3H2O that has been structurally and magnetically characterized. Magnetic studies on the Fe(II) complexes V and VI indicate that they undergo incomplete spin crossover transitions in the solid state. Strategies for the preparation of chiral spin crossover N3O2 macrocycles are discussed and the synthesis of the novel chiral Fe(II) macrocyclic complex VII is reported. Magnetic susceptibility and Mössbauer studies reveal that this complex undergoes a gradual spin crossover in the solid state with no thermal hysteresis. Variable temperature X-ray diffraction studies on single crystals of VII reveal interesting structural changes in the coordination geometry of the macrocycle accompanying its SCO transition. The second project reports the synthesis and characterization of a new family of tetrathiafulvalene derivatives VIII – XII, where a heterocyclic chelating ligand is appended to a TTF donor via an imine linker. The coordination chemistries of these ligands with M(hfac)2.H2O (M( = Co, Ni, Mn, Cu) have been explored and the structural and magnetic properties of these complexes are described.

Ligand Design for Dual Property Single Molecule Magnets

This thesis describes two different approaches for the preparation of polynuclear clusters with interesting structural, magnetic and optical properties. Firstly, exploiting p-tert-butylcalix[4]arene (TBC4) macrocycles together with selected Ln(III) ions for the assembly of emissive single molecule magnets, and secondly the preparation and coordination of a chiral mpmH ligand with selected 3d transition metal ions, working towards the discovery of chiral polynuclear clusters. In Project 1, the coordination chemistry of the TBC4 macrocycle together with Dy(III) and Tb(III) afforded two Ln6[TBC4]2 complexes that have been structurally, magnetically and optically characterized. X-ray diffraction studies reveal that both complexes contain an octahedral core of Ln6 ions capped by two fully deprotonated TBC4 macrocycles. Although the unit cells of the two complexes are very similar, the coordination geometries of their Ln(III) ions are subtly different. Variable temperature ac magnetic susceptibility studies reveal that both complexes display single molecule magnet (SMM) behaviour in zero dc field and the energy barriers and associated pre-exponential factors for each relaxation process have been determined. Low temperature solid state photoluminescence studies reveal that both complexes are emissive; however, the f-f transitions within the Dy6 complex were masked by broad emissions from the TBC4 ligand. In contrast, the Tb(III) complex displayed green emission with the spectrum comprising four sharp bands corresponding to 5D4 → 7FJ transitions (where J = 3, 4, 5 and 6), highlighting that energy transfer from the TBC4 macrocycle to the Tb(III) ion is more effective than to Dy. Examples of zero field Tb(III) SMMs are scarce in the chemical literature and the Tb6[TBC4]2 complex represents the first example of a Tb(III) dual property SMM assembled from a p-tert-butylcalix[4]arene macrocycle with two magnetically derived energy barriers, Ueff of 79 and 63 K. In Project 2, the coordination of both enantiomers of the chiral ligand, α-methyl-2-pyridinemethanol (mpmH) to Ni(II) and Co(II) afforded three polynuclear clusters that have been structurally and magnetically characterized. The first complex, a Ni4 cluster of stoichiometry [Ni4(O2CCMe3)4(mpm)4]·H2O crystallizes in a distorted cubane topology that is well known in Ni(II) cluster chemistry. The final two Co(II) complexes crystallize as a linear mixed valence trimer with stoichiometry [Co3(mpm)6]·(ClO4)2, and a Co4 mixed valence complex [Co(II)¬2Co(III)2(NO3)2(μ-mpm)4(ONO2)2], whose structural topology resembles that of a defective double cubane. All three complexes crystallize in chiral space groups and circular dichroism experiments further confirm that the chirality of the ligand has been transferred to the respective coordination complex. Magnetic susceptibility studies reveal that for all three complexes, there are competing ferro- and antiferromagnetic exchange interactions. The [Co(II)¬2Co(III)2(NO3)2(μ-mpm)4(ONO2)2] complex represents the first example of a chiral mixed valence Co4 cluster with a defective double cubane topology.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL), for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis. Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification / detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL capture is at least twice that of control, untreated sample.

Combinatorial peptide ligand libraries and plant proteomics: a winning strategy at a price

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides (combinatorial peptide ligand libraries or CPLL, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins Constituting the vast majority of species in any proteome. as applied to plant tissues, are reviewed here. Plant tissues are notoriously recalcitrant to protein extraction and to proteome analysis, Firstly, rigid plant cell walls need to be mechanically disrupted to release the cell content and, in addition to their poor protein yield, plant tissues are rich in proteases and oxidative enzymes, contain phenolic Compounds, starches, oils, pigments and secondary metabolites that massively contaminate protein extracts. In addition, complex matrices of polysaccharides, including large amount of anionic pectins, are present. All these species compete with the binding of proteins to the CPLL beads, impeding proper capture and identification I detection of low-abundance species. When properly pre-treated, plant tissue extracts are amenable to capture by the CPLL beads revealing thus many new species among them low-abundance proteins. Examples are given on the treatment of leaf proteins, of corn seed extracts and of exudate proteins (latex from Hevea brasiliensis). In all cases, the detection of unique gene products via CPLL Capture is at least twice that of control, untreated sample. (c) 2008 Elsevier B.V. All rights reserved.

Synthesis and evaluation of lipophilic BTBP ligands for An/Ln separation in nuclear waste treatment: the effect of alkyl substitution on extraction properties and implications for ligand design

Four new 6,6′-bis(1,2,4-triazin-3-yl)-2,2′-bipyridine (BTBP) ligands, which contain either additional alkyl groups on the pyridine rings or seven-membered aliphatic rings attached to the triazine rings, have been synthesized, and the effects of the additional alkyl substitution in the 4- and 4′-positions of the pyridine rings on their extraction properties with LnIII and AnIII cations in simulated nuclear waste solutions have been studied. The speciation of ligand 13 with some trivalent lanthanide nitrates was elucidated by 1H NMR spectroscopic titrations and ESI-MS. Although 13 formed both 1:1 and 1:2 complexes with LaIII and YIII, only 1:2 complexes were observed with EuIII and CeIII. Quite unexpectedly, both alkyl-substituted ligands 12 and 13 showed lower solubilities in certain diluents than the unsubstituted ligand CyMe4-BTBP. Compared to CyMe4-BTBP, alkyl-substitution was found to decrease the rates of metal-ion extraction of the ligands in both 1-octanol and cyclohexanone. A highly efficient (DAm > 10) and selective (SFAm/Eu > 90) extraction was observed for 12 and 13 in cyclohexanone and for 13 in 1-octanol in the presence of a phase-transfer agent. The implications of these results for the design of improved extractants for radioactive waste treatment are discussed.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA+CD27−CCR7−). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer+ T cells, confirming the role of CD8 T cells in this treatment strategy.

Transloading of tumor cells with foreign major histocompatibility complex class I peptide ligand: a novel general strategy for the generation of potent cancer vaccines.

The major hurdle to be cleared in active immunotherapy of cancer is the poor immunogenicity of cancer cells. In previous attempts to overcome this problem, whole tumor cells have been used as vaccines, either admixed with adjuvant(s) or genetically engineered to express nonself proteins or immunomodulatory factors before application. We have developed a novel approach to generate an immunogeneic, highly effective vaccine: major histocompatibility complex (MHC) class I-positive cancer cells are administered together with MHC class I-matched peptide ligands of foreign, nonself origin, generated by a procedure we term transloading. Murine tumor lines of the H2-Kd or the H2-Db haplotype, melanoma M-3 and B16-F10, respectively, as well as colon carcinoma CT-26 (H2-Kd), were transloaded with MHC-matched influenza virus-derived peptides and applied as irradiated vaccines. Mice bearing a deposit of live M-3 melanoma cells were efficiently cured by this treatment. In the CT-26 colon carcinoma and the B16-F10 melanoma, high efficacies were obtained against tumor challenge, suggesting the universal applicability of this new type of vaccine. With foreign peptide ligands adapted to the requirements of a desired MHC class I haplotype, this concept may be used for the treatment of human cancers.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Peptides constructed from α-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to "biomimics" of the DNA binding domain of LacI. Equilibrium dissociation constants (K D), association (k a), and dissociation rates (k d) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10 -10 M, 7.2 ± 0.6 × 10 -10 M, 4.5 ± 0.5 × 10 -8 M, and 6.2 ± 0.9 × 10 -6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon-repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.