A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.

This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification

Selective transesterification mediated by lanthanum complexes in the copolymerisation of lactide and δ-valerolactone †

Poly(lactide-co-valerolactone) copolymers were prepared via the one-pot copolymerisation of rac-lactide or S, S-lactide with δ-valerolactone at ambient temperature, mediated by bis(dimethylsilyl)amido lanthanum complexes supported by ligands derived from a salan framework (salan = N, N′-bis(o-hydroxy, m-di-tert-butylbenzyl)-1,2-diaminoethane), which incorporate either benzyl or 2-pyridyl groups at the tertiary amine moieties. Poly(δ-valerolactone)s were also prepared by the ring-opening polymerisation of δ-valerolactone and high molecular weight polymers (up to 83.6 kg mol−1) with narrow dispersities were obtained. The poly(lactide-co-valerolactone) copolymers were fully characterized using 1H and 13C NMR spectroscopy, gel-permeation chromatography and differential scanning calorimetry. Both the reaction solvent (toluene or THF) and the number of 2-pyridyl groups the complex possesses affect the complex activity and copolymer microstructure. The use of a non-coordinating solvent and presence of at least one 2-pyridyl group is required for high conversion of both monomers. Variation of the monomer feed ratio allowed copolymers across the full compositional range to be prepared. The copolymers are formed via a transesterification mechanism whereby all of the lactide undergoes rapid polymerisation in the early stages of the reaction and the δ-valerolactone is subsequently incorporated into the polymer. The rate and extent of δ-valerolactone polymerisation increases with the number of 2-pyridyl groups in the catalyst in toluene and is more rapid in non-coordinating solvent (toluene) than coordinating solvent (THF). Only low levels of the TII mode of transesterification occur, with the TI transesterification mode dominating, leading to the formation of copolymers with intact lactidyl units

Comparison of the structure and activity of glycosylated and asglycosylated human carboxylesterase 1

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme

Electrochemically Enhanced Drug Delivery Using Polypyrrole Films

The delivery of drugs in a controllable fashion is a topic of intense research activity in both academia and industry because of its impact in healthcare. Implantable electronic interfaces for the body have great potential for positive economic, health, and societal impacts; however, the implantation of such interfaces results in inflammatory responses due to a mechanical mismatch between the inorganic substrate and soft tissue, and also results in the potential for microbial infection during complex surgical procedures. Here, we report the use of conducting polypyrrole (PPY)-based coatings loaded with clinically relevant drugs (either an anti-inflammatory, dexamethasone phosphate (DMP), or an antibiotic, meropenem (MER)). The films were characterized and were shown to enhance the delivery of the drugs upon the application of an electrochemical stimulus in vitro, by circa (ca.) 10–30% relative to the passive release from non-stimulated samples. Interestingly, the loading and release of the drugs was correlated with the physical descriptors of the drugs. In the long term, such materials have the potential for application to the surfaces of medical devices to diminish adverse reactions to their implantation in vivo

The Development Of Novel Bio-based Polymeric Materials For Insulating Foams

This thesis details the synthesis of a series of novel thermosetting resins incorporating bio-based raw materials, with a view to their application to produce insulating foams. The work focuses primarily on phenolic resins/foams, and herein is reported a novel method for the synthesis of phenolic resin analogues. This method was employed in the synthesis of five novel thermosetting resins, which were formaldehyde-free and produced (partly) from bio-based raw materials. Initial investigations focused on the use of glyoxylic acid as a bio-based formaldehyde replacement in phenolic resin production. The properties of the resin obtained could be easily tailored to yield resins suitable for a variety of applications, including foaming. This resin was then blended with a phenolic resin to produce novel foams, which showed promising properties for application to insulating foams. Research then focused on the use of cardanol as a bio-based phenol for phenolic resin production, with a view to improving the mechanical properties of the resulting phenolic foams. The novel method was employed to produce four novel resins consisting of condensation products of cardanol and naturally occurring aldehydes. These resins could be cured to yield flexible polymers. However, the curing of these resins was poor, taking multiple days at elevated temperature to produce the thermoset polymers, making foaming difficult. The focus of the investigations then adjusted, to focus on a cardanol-formaldehyde resin, which exhibits superior curing to the novel resins. The first reports of foam production from cardanol-formaldehyde resins are then presented. Additionally, the incorporation of a series of aromatic species based on succinic acid into thermosetting resins is discussed. These investigations focused on phenolic resins and epoxy resins, the latter proving most successful

Structure–Property Relationships in Auxetic Liquid Crystal Elastomers—The Effect of Spacer Length

Auxetics are materials displaying a negative Poisson’s ratio, i.e., getting thicker in one or both transverse axes when subject to strain. In 2018, liquid crystal elastomers (LCEs) displaying auxetic behaviour, achieved via a biaxial reorientation, were first reported. Studies have since focused on determining the physics underpinning the auxetic response, with investigations into structure–property relationships within these systems so far overlooked. Herein, we report the first structure–property relationships in auxetic LCEs, examining the effect of changes to the length of the spacer chain. We demonstrate that for LCEs with between six and four carbons in the spacer, an auxetic response is observed, with the threshold strain required to achieve this response varying from 56% (six carbon spacers) to 81% (four carbon spacers). We also demonstrate that Poisson’s ratios as low as −1.3 can be achieved. Further, we report that the LCEs display smectic phases with spacers of seven or more carbons; the resulting internal constraints cause low strains at failure, preventing an auxetic response. We also investigate the dependence of the auxetic threshold on the dynamics of the samples, finding that when accounting for the glass transition temperature of the LCEs, the auxetic thresholds converge around 56%, regardless of spacer length

High-throughput cloning, expression, and purification

High-throughput sequencing of eukaryotic, viral, and bacterial genomes provides a huge database of proteins with potential for structure-function analysis. In response to this opportunity, structural genomics projects have been initiated world-wide with the aim of establishing high-throughput structure determination on a genome-wide scale. Crucial to this effort has been the development of protein production technologies for the highthroughput cloning, expression, and purification of proteins. Large-scale structural genomic projects were initiated in the US and Europe, and all have emphasized parallel processing, both in terms of molecular cloning, expression, and purification, driven by the need to accommodate relatively large numbers of potential targets for structural biology at an acceptable cost. This has led to varying degrees of automation and most of the groups involved have set up semiautomated liquid handling systems to carry out some or all of their protocols. However, the protocols can equally well be carried out manually with appropriate equipment, for example multichannel pipette dispensers. The motivation to implement automation is largely to enable processes to be scaleable and sustainable as error-free operations. This chapter reviews the technical developments that have come from structural proteomics and provides protocols for carrying out cloning, expression, and purification procedures in a relatively high-throughput (HTP) and parallel approach

High-throughput cloning, expression, and purification

High-throughput sequencing of eukaryotic, viral, and bacterial genomes provides a huge database of proteins with potential for structure-function analysis. In response to this opportunity, structural genomics projects have been initiated world-wide with the aim of establishing high-throughput structure determination on a genome-wide scale. Crucial to this effort has been the development of protein production technologies for the highthroughput cloning, expression, and purification of proteins. Large-scale structural genomic projects were initiated in the US and Europe, and all have emphasized parallel processing, both in terms of molecular cloning, expression, and purification, driven by the need to accommodate relatively large numbers of potential targets for structural biology at an acceptable cost. This has led to varying degrees of automation and most of the groups involved have set up semiautomated liquid handling systems to carry out some or all of their protocols. However, the protocols can equally well be carried out manually with appropriate equipment, for example multichannel pipette dispensers. The motivation to implement automation is largely to enable processes to be scaleable and sustainable as error-free operations. This chapter reviews the technical developments that have come from structural proteomics and provides protocols for carrying out cloning, expression, and purification procedures in a relatively high-throughput (HTP) and parallel approach