Modification of bacterial microcompartments with target biomolecules post-translational SpyTagging.

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein.

Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription.

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear functions for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52.

Optimal His-Tag Design for Efficient [Tc(CO)] and [Re(CO)] Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [Tc(CO)] and [Re(CO)] for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [Tc(CO)] and [Re(CO)] to identify key features that enhance labeling.

The effect of caffeine on cognitive performance is influenced by CYP1A2 but not ADORA2A genotype, yet neither genotype affects exercise performance in healthy adults.

Purpose: To determine the influence of two commonly occurring genetic polymorphisms on exercise, cognitive performance, and caffeine metabolism, after caffeine ingestion.

Methods: Eighteen adults received caffeine or placebo (3 mg kg) in a randomised crossover study, with measures of endurance exercise (15-min cycling time trial; 70-min post-supplementation) and cognitive performance (psychomotor vigilance test; PVT; pre, 50 and 95-min post-supplementation). Serum caffeine and paraxanthine were measured (pre, 30 and 120-min post-supplementation), and polymorphisms in ADORA2A (rs5751876) and CYP1A2 (rs762551) genes analysed.

Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over.

[Re(CO)(3)](+) labelling of a novel cysteine/hexahistidine tag: insights into binding mode by liquid chromatography-mass spectrometry.

We recently described a novel amino acid sequence, KCKLAAALEHHHHHH, for site-specific radiolabelling of proteins with [(99m)Tc(CO)(3)(OH(2))(3)](+) or [Re(CO)(3)(OH(2))(3)](+) with improved efficiency compared to conventional hexahistidine tags (His-tag). C2AH, a modification of the protein C2A (the phosphatidylserine (PS)-binding domain of rat synaptotagmin I) engineered to contain this novel C-terminal tag, was produced. Rhenium tricarbonyl conjugates of C2AH were analysed post tryptic digest by liquid chromatography-electrospray mass spectrometry (LC-MS), giving rise to a peak with the molecular weight corresponding to M(+)=[Re(CO)(3)+CK+LAAALEHHHHHH](+).

A novel HPLC method for the concurrent analysis and quantitation of seven water-soluble vitamins in biological fluids (plasma and urine): a validation study and application.

An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B(1), B(2), B(5), B(6), B(9), B(12)) in biological matrices (plasma and urine). Separation was achieved at 30°C on a reversed-phase C18-A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol.

Formation of a solubilized cobalt block oligomer from a M2L-type double helicate.

The multi-dentate ligand, 2,3,5,6-tetrakis(2,2'-bipyridyl)pyrazine (L) and divalent cobalt self-assemble to a block co-polymer-like oligomer in solution, which contains at least the L(7)Co(8) fragment. The extent of oligomerization is sensitive to the water content in acetonitrile solution. In the solid state, the simple monomer [LCo(2)(CH(3)CN)(4)][ClO(4)](4) is isolated.

Solid-phase synthesis of peptide radiopharmaceuticals using Fmoc-N-epsilon-(hynic-Boc)-lysine, a technetium-binding amino acid: application to Tc-99m-labeled salmon calcitonin.

Labeling of proteins with metallic radionuclides for use in radiopharmaceuticals involves covalently attaching a bifunctional chelator. In principle, use of smaller peptides allows this chelator to be incorporated during solid-phase peptide synthesis (SPPS) with total site specificity. To realize the advantages of this approach, a lysine-hynic conjugate Fmoc-N-epsilon-(Hynic-Boc)-Lys was synthesized for incorporating the well-known technetium-99m-binding hydrazinonicotinamide ligand into peptides during SPPS.

Analysis of a retan agent used in the tanning process and its determination in tannery wastewater.

The chemical structure and composition of a retan agent, CNSF (condensation product of naphthalenesulfonic acid (NSA) and formaldehyde), and related components contained in tannery wastewaters were analyzed by ion-pair liquid chromatography coupled to electrospray ionization mass spectrometry (IPC-HPLC/ESI-MS) in negative ion mode. This method allows high-resolution separation of polymers. CNSF contained linear NSA oligomers (n = 1-11) that were eluted in order of increasing degree of polymerization.