Production of lectin-affinity matrices for process-scale glycoprotein purification

A selection of prokaryotic lectins with a variety of glycan specificities and affinities have been identified, cloned, expressed in Eschericia coli and characterised. The aims of this project are to: - express the lectins at 1L scale to produce sufficient quantities for immobilisation studies (~100 mg) - immobilisethelectinsonSepharose - evaluate lectin performance on column by monitoring their ability toreproducibly capture and elute glycoprotein glycoforms

Effect of Loss on Multiplexed Single-Photon Sources

An on-demand single-photon source is a key requirement for scaling many optical quantum technologies. A promising approach to realize an on-demand single-photon source is to multiplex an array of heralded single-photon sources using an active optical switching network. However, the performance of multiplexed sources is degraded by photon loss in the optical components and the non-unit detection efficiency of the heralding detectors. We provide a theoretical description of a general multiplexed single-photon source with lossy components and derive expressions for the output probabilities of single-photon emission and multi-photon contamination. We apply these expressions to three specific multiplexing source architectures and consider their tradeoffs in design and performance. To assess the effect of lossy components on near- and long-term experimental goals, we simulate the multiplexed sources when used for many-photon state generation under various amounts of component loss. We find that with a multiplexed source composed of switches with ~0.2-0.4 dB loss and high efficiency number-resolving detectors, a single-photon source capable of efficiently producing 20-40 photon states with low multi-photon contamination is possible, offering the possibility of unlocking new classes of experiments and technologies.Comment: Journal versio

Genetically enhanced recombinant lectins for glyco-selective analysis and purification

- Generation of a library of recombinant prokaryotic lectins (RPL’s) through random mutagenesis of the carbohydrate binding sites of bacterial lectins. - Characterisation of mutant lectins with respect to structure and specificity - Provision of mutant RPL’s with enhanced affinity and/or altered specificity, alongside wild-type RPL’s, for glycoprotein analysis and purificatio

The investigation of a recombinant GalNAc binding protein from bacillus thuringiensis as a tool for glycan analysis and detection

Changes in the structures of glycans on the surfaces of eukaryotic cells can be important biomarkers for developmental or disease states. Improved methods are needed for the detection and analysis of alterations in glycan structures. Carbohydrate binding proteins such as lectins have potential for the recognition of changes in glycan structure. Host-pathogen interactions frequently involve the recognition of host carbohydrates by proteins of bacteria or viruses. Many bacterial toxins have evolved to interact with host cell receptors or with a specific tissue due to lectin like properties. The toxins from Bacillus thuringiensis have been shown to have carbohydrate binding abilities, in particular N-Acetylgalactosamine (GalNAc) has been shown to inhibit the binding of the toxin Cry1Ac. GalNAc has been shown to be an important marker in many diseases such as breast cancer and colon carcinogenesis. Moreover, changes in GalNAc glycosylation have been identified in many disorders such as cystic fibrosis, neuromuscular disorders and nephropathy. Here we describe the purification of a GalNAc binding protein of bacterial origin that may have potential in the development of diagnostic assays

Exploitation of siderophores for the speciation of iron

Iron is essential for life. It acts as an electron donor/acceptor in metabolic processes facilitated by its variable valency. Although vital, it is toxic at high levels due to Fe2+ oxidation. Iron toxicity is a concern as it can affect growth and product yields in animal cell culture. Siderophores are high affinity Fe3+ chelators produced by microorganisms. This affinity gives them the potential to be used as a basis in platforms to detect and speciate iron in industrial cell culture. Rhizobactin 1021 is of interest due to its decanoic acid “tail” that is not involved in chelation which makes it an ideal target for immobilisation

Regions of the Cry1Ac toxin predicted to be under positive selection are shown to be the carbohydrate binding sites and can be altered in their glycoprotein target specificity

The cry gene family, is a large family of homologous genes from Bacillus thuringiensis. Studies have examined the structural and functional relationships of the Cry proteins. They have revealed several residues in domains II and III that are important for target recognition and receptor attachment. In 2007 Wu, Jin-Yu et al employed a maximum likelihood method to detect evidence of adaptive evolution in Cry proteins. They identified positively selected residues, which are all located in Domain II or III. Figure 1 shows a protein sequence alignment between domain II and III of Cry1Ac and Cry1Aa. This highlights the areas which are thought to be under positive selection. Cry1Ac and Cry1Aa are structurally very similar and they both bind to a variety of N-aminopeptidases (APN’s) in different insect species. However Cry1Aa has a higher specificity for the cadherin like receptor HevCalP and Cry1Ac binds to N-acetylgalactosamine (GalNAc) on the surface of APN’s. Differences in the binding of the two toxins has been shown in an in-direct toxin-binding assay where GalNAc completely abolished toxin binding of Cry1Ac but had no effect on the binding of Cry1Aa. The binding site has been shown to be located in the third domain of Cry1Ac. Some of these sites correlate with the positively selected residues found by Wu et al 2007 in Cry1Aa. Our aim was to use the comparison of the toxins to analyse the potential to alter the binding specificity of Cry1Ac and its domains. In this work we identified critical amino acid residues for this objective

Electronic structure calculations and physicochemical experiments quantify the competitive liquid ion association and probe stabilisation effects for nitrobenzospiropyran in phosphonium-based ionic liquids.

Liquid ion association in ionic liquids (ILs) has been examined using a comprehensive series of electronic structure calculations that measure the relative extents of ion association and probe stabilisation for the photochromic dye nitrobenzospiropyran (BSP) in a range of ILs featuring both long-tailed phosphonium cations and short-tailed imidazolium cations, paired with both chloride and NTf2 anions. New physicochemical experiments measured the photochromic properties of BSP in the phosphonium-based room temperature ILs. Taken together, the computed complexation energies and measured spectroscopic properties support recent Walden plots of unusual conductivity–viscosity behaviour obtained for the same ILs and reveal some new features in the atom-scale structure and energetics of local, ion–ion and ion–molecule interactions. Calculations show inter-ion interactions strengthened by between 0.4 and 0.7 eV as stronger constituent ions are used, which contributes to the longer range rigidity of the Cl-based IL structure as reflected in the doubled |zwitterion - closed| probe relaxation time measured for Cl vs. NTf2 in phosphonium-based ILs. Calculations further reveal a similar, approximately 0.6–0.7 eV maximum ‘‘residual’’ IL headgroup-mediated probe stabilisation potentially available for the anion–probe–cation complexes via the stabilising interaction that remains following the ‘‘quenching’’ interaction between the IL anion and cation. This potential stabilisation, however, is offset by both longer-range charge networks, beyond the scope of the current purely quantum mechanical simulations, and also energetic penalties for disruption of the highly-interdigitated alkyl tail networks in the phosphonium-based ILs which may be estimated from known diffusion data. Overall the electronic calculations of local, individual ion–ion and ion–molecule interactions serve to clarify some of the measured physicochemical properties and provide new data for the development of classical force field-based approaches to measure also the longer range effects that, together with the electronic effects, provide the condensed phase IL structure and properties. More generally, the combined simulation and experimental results serve as a further example of how both the polar hydrophilic headgroup and non-polar hydrophobic tail of the constituent ions serve as distinct targets for IL rational design